Use of Candida bombicola strains modified in their sophorolipid production

ABSTRACT

The present invention relates to yeast species which are normally capable of producing sophorolipids but which are modified in such way that they are incapable producing the latter compounds. These sophorolipid-negative strains surprisingly display equal growth characteristics and biomass formation as their wild type counterparts and are hence useful for the production of compounds such as recombinant proteins, glycolipids, polyhydroxyalkanoates and carotenoides. In addition, the present invention discloses two glucosyltransferase genes with key-functions in sophorolipid production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/EP2011/059683, filed Jun. 10, 2011, whichclaims priority to GB 1009882.0, filed Jun. 11, 2010.

REFERENCE TO SEQUENCE LISTING

This application incorporates by reference the sequence listingsubmitted as ASCII text filed via EFS-Web on Jan. 13, 2015. The SequenceListing is provided as a file entitled “seqlstUSdecle176004apc.txt,”created on Jan. 8, 2015, and which is approximately 38 kilobytes insize.

TECHNICAL FIELD OF INVENTION

The present invention relates to yeast species which are normallycapable of producing sophorolipids but which are so modified that theybecome incapable of producing the latter compounds. Thesesophorolipid-negative strains surprisingly display equal growthcharacteristics and biomass formation as their wild type counterpartsand are hence useful for the production of numerous useful compoundssuch as recombinant proteins, glycolipids, polyhydroxyalkanoates,special sugars, special fatty acids, squaleen, organic acids,hydrophobic compounds, and carotenoides. In addition, the presentinvention discloses two glucosyltransferase genes with key-functions insophorolipid production and their use.

BACKGROUND ART

The non-pathogenic yeast Candida bombicola and other yeast species suchas Candida apicola, Candida batistae, Rhodotorula bogoriensis andWickerhamiella domericqiae are known for their sophorolipid productionduring stationary phase (Spencer et al., 1970, Gorin et al., 1961,Tulloch et al., 1968, EP0837140A1, U.S. Pat. No. 6,433,152, U.S. Pat.No. 4,215,213). C. bombicola and others are oleaginous yeast species,i.e. they can utilize oleaginous substrates such as alkanes and oils ascarbon source, and can handle those substrates in relatively highconcentrations. Moreover, C. bombicola can produce sophorolipids in highamounts (over 400 g/L), which are excreted in the fermentation medium.

Candida bombicola ATCC 22214 is already applied commercially for theproduction of sophorolipids. These glycolipid biosurfactants areconstituted of a sophorose head group(2-O-β-D-glucopyranosyl-β-D-glucopyranose) from which the anomericC-atom is attached to an (ω) or (ω-1) C₁₆ or C₁₈ hydroxylated fattyacid. They occur either as open-ring structures (acid form) or aslactones with an intra-esterification between the fatty acid carboxylgroup and the 4″, 6′ or 6″ carbon atom of the sophorose head group. Inaddition, acetyl groups can be attached at the 6′ and/or 6″ positions(Asmer et al., 1988).

In a typical Candida bombicola fermentation with e.g. rapeseed oil as ahydrophobic carbon source, sophorolipids are present as a complexmixture of structurally related molecules with the mono- anddi-acetylated lactonic sophorolipids being the most important.

While many research has focused on fermentation conditions to optimizesophorolipid production by C. bombicola (Daniel et al., 1998a, Daniel etal., 1998b, Casas et al., 1999, Cavalero et al., 2003, Kim et al.,2009), and sophorolipids have served as substrates for (chemo)-enzymaticmodifications (Bisht et al., 1999, Hu et al., 2003, Rau et al., 1999),the biochemical pathway of these economically important bioproductsremains unclear and there is no information available about the genesinvolved (Ochsner et al., 1994a, Ochsner et al., 1994b). This lack ofinformation hampers implementation of modern techniques such asmetabolic engineering to increase sophorolipid yields. The only dataabout enzymes involved in sophorolipid production by C. bombicolasuggest the involvement of a cytochrome P450 monooxygenase from theCYP52 family (Van Bogaert et al., 2009). Data about enzymes in otheryeasts are limited to protein experiments with C. bogoriensis lysatesand date from early 1970's and 1980's (Esders et al., 1972, Bucholtz etal., 1976, Breithaupt et al., 1982). Apart from an acetyltransferase, itwas assumed that two glucosyltransferases were involved in the stepwiseproduction of sophorolipid by this organism, though separation of thetwo activities remained unsuccessful (Breithaupt et al., 1982).

Besides the fact that the biochemical pathway of the production ofsophorolipids is largely unknown, C. bombicola will always producesophorolipids no matter which carbon source is applied. Hence, thesophorolipids will always form a mixture with other compounds ofpotential interest and the biosynthetic pathway will compete for the useof substrates. Consequently, it is hardly impossible to combine the(recombinant) production of a compound of interest with the sophorolipidsynthesis without substantial loss of efficiency and without additionalpurification costs. The option to only produce the compound of interestduring the exponential growth phase when no or low amounts ofsophorolipids are produced, will not only result in lower yields due tothe lower amount of available biomass and shorter production time, butwill also often result in a low tolerance of the growing cells towardsthe oleaginous substrates used. These problems are omitted when strainsthat lack this sophorolipid production are used. However, Ito and Inoue(Ito et al., 1982; Inoue et al., 1982) demonstrated that sophorolipidsstimulate the growth on oleaginous substrates whereas a number ofsynthetic non-ionic surfactants have no effect. They further state thatsophorolipids act as specific growth stimulating factors and are neededto emulsify the insoluble oleaginous substrates. Therefore, strainsunable to produce sophorolipids show inferior growth as compared to thewild type and are unable to handle oleaginous substrates.

Taken together, it is clear that sophorolipid-producing yeast speciesmight be very useful to produce, in addition to sophorolipids, othernumerous useful compounds such as recombinant proteins, glycolipids,polyhydroxyalkanoates, sophorose, rhamnose, special fatty acids,squaleen, organic acids, hydrophobic compounds and oleageniouscompounds. However, a lack of understanding of the underlyingbiochemical pathway of sophorolipid synthesis and the problem ofcombining the production of a useful compound of interest with thesophorolipid synthesis without substantial loss of efficiency andwithout additional purification costs seriously hamper the usage ofthese yeast species.

Description of Tables:

-   Table 1 Primers used for knocking-out the C. bombicola CYP52M1 gene.    All primers were obtained from Sigma Genosys.-   Table 2 Primers used for isolation of the UGTA1 gene and    construction of the knock-out cassette. All primers were obtained    from Sigma Genosys.-   Table 3 Ten best homology scores for the translated UGTA1 sequence-   Table 4 Primers used for isolation of the UGTB1 gene and    construction of the knock-out cassette. All primers were obtained    from Sigma Genosys.-   Table 5 Ten best homology scores for the translated UGTB1 sequence-   Table 6 Primers used for creation of the GFP expression cassette.    All primers were obtained from Sigma Genosys. Bold characters    represent non-binding extensions.-   Table 7 Primers used for creating the amylase expression cassette    and control of amylase transformants.-   Table 8 Primers used for creating the PHA expression cassette. All    primers were obtained from Sigma Genosys.-   Table 9 Primers used for creation of the UGT1 and CepB expression    cassettes. All primers were obtained from Sigma Genosys.

DESCRIPTION OF FIGURES

FIG. 1 HPLC-ELSD analysis of the sophorolipid production medium forCandida bombicola wild-type (up) and one of the cyp52M1-negative strains(down). Major class sophorolipids are detected between 25 and 30 min.Oleic acid and linoleic acid elute at 33.5 and 36.2 min respectively.

FIG. 2 Growth of C. bombicola wildtype, A113 (ugtA1 deletion mutant) andB11 (ugtB1 deletion mutant) in Lang medium. All cultures were inoculatedfrom a Lang preculture in such a way that all the cultures started withOD 0.2.

FIG. 3 Complete DNA sequence of the UGTA1 gene (GenBank accession numberHM440973, SEQ ID NO° 1). Putative promoter and terminator elements areindicated by boxes while a possible GATA-like regulatory motif is shadedin grey. The encoded amino acid sequence (SEQ ID NO° 2) of the Ugta1glucosyltransferase protein is also depicted.

FIG. 4 HPLC-ELSD chromatograms from culture extracts, 10 days afterincubation of C. bombicola ATCC22214 (up) and C. bombicola A113 (down)with rapeseed oil. De novo sophorolipids typically elute between 25 and30 min.

FIG. 5 HPLC-ELSD chromatogram of sample extracts from aglucosyltransferase I assay on soluble protein fractions from wildtypeyeast (up) and A113 mutant (down). Peaks at 27 min and 29 min originatefrom de novo sophorolipid synthesis while peaks eluting after 30 minutesoriginate from co-extracted apolar cell constituents. FA 17-OHC18:1=17-hydroxyoctadecenoic acid, GL=glucolipid(17-O-(β-D-glucopyranosyl)-octadecenoic acid), SL=diacetylatedsophorolipid acid(17-O-(2-O-β-D-glucopyranosyl-glucopyranose)-octadecenoic acid), lacSL2Ac=diacetylated sophorolipid lactone

FIG. 6 HPLC-ELSD chromatogram of sample extracts from aglucosyltransferase II assay on soluble protein fractions from wildtypeyeast (up) and A113 mutant (down). Peaks at 27 min and 29 min originatefrom de novo sophorolipid synthesis while peaks eluting after 30 minoriginate from co-extracted apolar cell constituents. FA 17-OHC18:1=17-hydroxyoctadecenoic acid, GL=glucolipid(17-O-(β-D-glucopyranosyl)-octadecenoic acid), SL=diacetylatedsophorolipid acid(17-O-(2-O-β-D-glucopyranosyl-glucopyranose)-octadecenoic acid), lacSL2Ac=diacetylated sophorolipid lactone

FIG. 7 Complete DNA sequence of the UGTB1 gene (GenBank accession numberHM440974, SEQ ID NO° 3). Primer sites are underlined. Putative promoterand terminator elements are indicated by boxes while possible GATA-likeregulatory motifs are shaded in grey. The encoded amino acid sequence(SEQ ID NO° 4) of the UgtB1 glucosyltransferase protein is alsodepicted.

FIG. 8 Global pairwise alignment of C. bombicola UgtA1 (upper; SEQ IDNO: 2) and UgtB1 (lower; SEQ ID NO: 4) protein sequences. Similar aminoacids are shaded in grey while identical residues are shaded in black.The 14 conserved residues of the GT1_Gtf_like domain are indicated byarrows down for UgtA1 and arrows up for UgtB1.

FIG. 9 HPLC-ELSD chromatograms from culture extracts, 7 days afterincubation of C. bombicola ATCC22214 (up) and C. bombicola B11 (down)with rapeseed oil. De novo sophorolipids typically elute between 25 and30 min. SL=sophorolipid, GL=glucolipid.

FIG. 10 HPLC-ELSD chromatogram of sample extracts from aglucosyltransferase I assay on soluble protein fractions from wildtypeyeast (up) and B11 mutant (down). Peaks at 27 min and 29 min come fromde novo sophorolipid synthesis while peaks eluting after 30 min comefrom co-extracted apolar cell constituents. FA 17-OHC18:1=17-hydroxyoctadecenoic acid, GL=glucolipid(17-O-(β-D-glucopyranosyl)-octadecenoic acid), SL=diacetylatedsophorolipid acid(17-O-(2-O-β-D-glucopyranosyl-glucopyranose)-octadecenoic acid), lacSL2Ac=diacetylated sophorolipid lactone

FIG. 11 HPLC-ELSD chromatogram of sample extracts from aglucosyltransferase II activity test on soluble protein fractions fromwildtype yeast (up) and B11 mutant (down). Peaks at 27 min and 29 mincome from de novo sophorolipid synthesis while peaks eluting after 30min come from co-extracted apolar cell constituents. FA 17-OHC18:1=17-hydroxyoctadecenoic acid, GL=glucolipid(17-O-(β-D-glucopyranosyl)-octadecenoic acid), SL=diacetylatedsophorolipid acid(17-O-(2-O-β-D-glucopyranosyl-glucopyranose)-octadecenoic acid), lacSL2Ac=diacetylated sophorolipid lactone

FIG. 12 Fluorescent signal in function of time for the wild type and theE1 GFP mutant

FIG. 13 Molecular map of the amylase synthetic construct, preceded bythe partial GAPD promoter

FIG. 14 Sequence of the amylase synthetic construct, preceded by part ofthe GAPD promoter (nucleic acid sequence is SEQ ID NO: 64; amino acidsequence is SEQ ID NO: 65).

FIG. 15 Vector p_sAmyAO_pGapd_iUra

FIG. 16 Amylase production by the sophorolipid negative amylasetransformant (SL− Amy+) and the wild type (WT) of C. bombicola

FIG. 17 Metabolic profile of the sophorolipid negative amylasetransformant (SL− Amy+, left) and the wild type (WT, right) of C.bombicola

FIG. 18 PHA expression cassette and its integration at the cyp52m1 locus

FIG. 19 GC-MS analysis of the FAMES derived from end samples of theCandida bombicola PHAC1 A8 mutant grown on Lang medium with addition ofrapeseed oil after 48 h of cultivation

FIG. 20 Mass spectra of the three predominant glucolipids as found in aB11 culture extract 14 days after cultivation on rapeseed oil (37.5g/L).

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates that, surprisingly,sophorolipid-negative strains displayed equal growth characteristics andbiomass formation as compared to the wild type and were not inhibited bythe presence of oleaginous substrates. The invention further disclosesthat sophorolipid-negative strains can be used to produce usefulcompounds. In addition, the present invention also discloses twoglucosyltransferase genes with key-functions in sophorolipid production.

Therefore, the invention relates to a modified yeast strain belonging toa yeast species capable of producing sophorolipids characterized in thatsaid yeast strain has, compared to an unmodified wild-type strain: a) atleast one mutation in its genome, and b) a reduction in its capabilityof producing sophorolipids of at least 75%, and wherein saidsophorolipids are constituted of the sugar sophorose attached to a C₁₆,C₁₈, C₂₂ or C₂₄ hydroxylated fatty acid. The reduction in saidcapability must be measured by fermentation of said mutant and saidwildtype strain under exactly the same fermentation conditions, andsimilarly, sophorolipid production by said mutant and said wildtype mustbe measured by exactly the same method. Quantification of saidsophorolipid production can—for example—be undertaken afterprecipitation from the fermentation medium or after extraction withethylacetate as described by Lang et al. (2000).

The term ‘yeast species capable of producing sophorolipids’ refers to aphylogenetically diverse group of yeasts (predominantly Ascomycetes andfew Basidiomycetes) which spontaneously synthesize sophorolipidsconstituted of the sugar sophorose attached to a hydroxylated fattyacid. Said phylogenetically diverse group of yeasts comprises thespecies Candida apicola (Gorin et al., 1961) which was initiallyidentified as C. magnolia, C. bombicola (Spencer et al., 1970),Wickerhamiella domericqiae (Chen et al., 2006), Rhodotorula bogoriensis(Tulloch et al., 1968), Pichia anomala PY1, Candida batistae (Konishi etal., 2008), Candida floricola (Imura et al., 2010), Candida riodocensis,Candida stellata and Candida sp. NRRL Y-27208 (Kurtzman et al., 2010)and other species of the so-called Starmerella clade which encorporatesover 40 species. C. bombicola has been recently reassigned to the genusStarmerella (Rosa & Lachance, 1998).

Therefore, the invention further relates to a modified yeast strain asindicated above wherein said yeast species is selected from the group ofsophorolipid producing yeasts, consisting of Candida bombicola, Candidaapicola, Candida batistae, Candida floricola, Candida riodocensis,Candida stellata, Candida sp. NRRL Y-27208, Rhodotorula bogoriensis,Pichia anomala PY1, Wickerhamiella domericqiae andsophorolipid-producing species of the Starmerella clade. Morespecifically, the invention relates to a modified yeast strain asindicated above wherein said Candida bombicola is the strain Candidabombicola ATCC 22214.

The term ‘modified yeast strain’ indicates that the genetic material ofsaid yeast strain has been altered so that yeast strain is incapable, oris almost incapable of producing sophorolipids. The term ‘almostincapable’ indicates that said modified yeast strain is solely capableof producing a maximum of 25% of the total production per time unit ofsophorolipids by a so called unmodified wild-type strain. Modified yeaststrains that are solely capable of producing a maximum of 20%, 15%, 10%or 5% of the total production per time unit of sophorolipids by a socalled unmodified wild-type strain are also envisioned by the presentinvention. Modified yeast strains that are completely incapable (0%) ofproducing sophorolipids are a preferred embodiment of the presentinvention. The term ‘unmodified wild-type strain’ refers to a yeaststrain as it occurs in nature and which is fully capable (100%) ofproducing sophorolipids.

More specifically, the term ‘modified yeast strain’ refers to a yeaststrain containing at least one mutation in its genome. The term mutationrefers to a spontaneous mutation and/or to an induced mutation in thegenome of said yeast strain. Said mutation can be a point mutation,deletion, insertion or any other type of mutation. The term mostspecifically refers to knock outs (KO) via insertion of a KO cassette.

Inducing a mutation in the genome of a yeast strain can be undertaken byany method in the art known by a skilled person such as the insertion ofa KO cassette into a gene of interest. Similarly, tracing or detectingwhether there is a mutation in the genome of a modified gene—compared toa wild type strain—can also be determined by any method known in theart. The term ‘sophorolipids’ refer to carbohydrate-based, amphiphilicbiosurfactants that are constituted of the sugar sophorose attached to aC₁₆, C₁₈, C₂₂ or C₂₄ hydroxylated fatty acid, i.e. hydroxylated fattyacids wherein the fatty acid chain is composed of 16, 18, 22 or 24C-atoms. More specifically the term refers to glycolipid biosurfactantsthat are constituted of a sophorose head group(2-O-β-D-glucopyranosyl-D-glucopyranose) from which the anomeric C-atomis attached to an (ω) or (ω-1) hydroxylated C₁₆, C₁₈, fatty acid or to aC₂₂ or C₂₄ fatty acid hydroxylated at the C₁₃ position. With regard toC. bombicola—said fatty acid chain is composed of 16 or 18 C-atoms. Theyoccur either as open-ring structures (acid form) or as lactones with anintra-esterification between the fatty acid carboxyl group and the, 4″,6′ or 6″ carbon atom of the sophorose head group. In addition, acetylgroups can be attached at the 6′ and/or 6″ positions (Asmer et al.,1988). In a typical Candida bombicola fermentation with e.g. rapeseedoil as the hydrophobic carbon source, sophorolipids are present as acomplex mixture of structurally related molecules with the mono- anddi-acetylated lactonic sophorolipids being the most important.

The invention further concerns a modified yeast strain as indicatedabove, wherein said mutation is a deletion in a gene encoding for aprotein, more specifically wherein said protein is an enzyme or aregulatory protein, involved in the sophorolipid biosynthetic pathway.More specifically, the invention relates to mutations in the genesCYP52M1 (Van Bogaert et al., 2009) encoding for a cytochrome P450monooxygenase, UGTA1 (depicted by SEQ ID No 1) encoding forglucosyltransferase 1 and UGTB1 (depicted by SEQ ID No 3) encoding forglucosyltransferase 2.

Hence, and more specifically the present invention relates to a modifiedyeast strain as indicated above wherein said gene encodes for acytochrome P450 monooxygenase or a glucosyltransferase, and morespecifically wherein said gene encoding for a cytochrome P450monooxygenase is the CYP52M1 gene having Genbank accession numberEU552419 and wherein said gene encoding for a glucosyltransferase is theUGTA1 gene having a sequence as depicted by SEQ ID No 1 and havingGenbank accession number HM440973 or is the UGTB1 gene having a sequenceas depicted by SEQ ID No 3 and having Genbank accession number HM440974.

The invention further relates to a nucleic acid sequence as depicted bySEQ ID No 1 encoding for the UDP-glucosyltransferase UgtA1 responsiblefor the first glucosylation step in the sophorolipid biosyntheticpathway of Candida bombicola, or a fragment thereof encoding for aprotein retaining said UDP-glucosyltransferase activity, or a variantthereof encoding for a protein having at least 50% sequence identitywith SEQ ID No 2 and having said UDP-glucosyltransferase activity. Theterm ‘fragment’ refers to a nucleic acid sequence containing fewernucleotides than the nucleic acid sequence as depicted by SEQ ID No 1and that encodes for a protein retaining said UDP-glucosyltransferaseactivity. The term “variant” refers to a nucleic acid encoding for aprotein having at least 50% sequence identity, preferably having atleast 51-70% sequence identity, more preferably having at least 71-90%sequence identity or most preferably having at least 91, 92, 93, 94, 95,96, 97, 98 or 99% sequence identity with SEQ ID No 2 or with a fragmentthereof, and, that encodes for a protein retaining saidUDP-glucosyltransferase activity.

The invention also relates to an amino acid sequence as depicted by SEQID No 2 and corresponding to the UDP-glucosyltransferase UgtA1responsible for the first glucosylation step in the sophorolipidbiosynthetic pathway of Candida bombicola, or a fragment thereof havingsaid UDP-glucosyltransferase activity or a variant thereof having atleast 50% sequence identity with SEQ ID No 2 and having saidUDP-glucosyltransferase activity. The term ‘fragment’ refers to aprotein containing fewer amino acids than the amino acid sequence asdepicted by SEQ ID No 2 and that retains said UDP-glucosyltransferaseactivity. Such fragment can for example omit N- and C-termini of theprotein leaving a shortened protein of 366 amino acids starting at 110and ending at A375 without hereby touching the UDP-glucosyltransferaseactivity. The term “variant” refers to a protein having at least 50%sequence identity, preferably having at least 51-70% sequence identity,more preferably having at least 71-90% sequence identity or mostpreferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99%sequence identity with SEQ ID No 2 or with a fragment thereof, and, thatencodes for a protein retaining said UDP-glucosyltransferase activity.The latter may differ from the protein as depicted by SEQ ID No 2 or afragment thereof only in conservative substitutions and/ormodifications, such that the ability of the protein to haveUDP-glucosyltransferase activity is retained. A “conservativesubstitution” is one in which an amino acid is substituted for anotheramino acid that has similar properties, such that one skilled in the artof protein chemistry would expect the nature of the protein to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be proteins as described hereinmodified by, for example, by the deletion or addition of amino acidsthat have minimal influence on the UDP-glucosyltransferase activity,secondary structure and hydropathic nature of the protein. Regionswithin SEQ ID No 2 which contribute to the proteins' activity areregions determined by the residues I10 to V50, G131 to L155, M180 toH196, W213 to F233, S266 to Y282, V341 to H355, A375 to T386. Hence, thevariants as defined above preferably comprise at least one of the latterregions. More preferably, the latter variants comprise at least one ofthe following residues or amino acids of SEQ ID No 2: G19, H20, M22,N181, L184, E185, F189, S216, T277, N343, G345, G347, G348 or H351.

The invention further relates to a nucleic acid sequence as depicted bySEQ ID No 3 encoding for the UDP-glucosyltransferase UgtB1 responsiblefor the second glucosylation step in the sophorolipid biosyntheticpathway of Candida bombicola, or a fragment thereof encoding for aprotein retaining said UDP-glucosyltransferase activity, or a variantthereof encoding for a protein having at least 50% sequence identitywith SEQ ID No 4 and having said UDP-glucosyltransferase activity. Theterm ‘fragment’ refers to a nucleic acid sequence containing fewernucleotides than the nucleic acid sequence as depicted by SEQ ID No 3and that encodes for a protein retaining said UDP-glucosyltransferaseactivity. The term “variant” refers to a nucleic acid encoding for aprotein having at least 50% sequence identity, preferably having atleast 51-70% sequence identity, more preferably having at least 71-90%sequence identity or most preferably having at least 91, 92, 93, 94, 95,96, 97, 98 or 99% sequence identity with SEQ ID No 4 or with a fragmentthereof, and, that encodes for a protein retaining saidUDP-glucosyltransferase activity.

The invention also relates to an amino acid sequence as depicted by SEQID No 4 and corresponding to the UDP-glucosyltransferase UgtB1responsible for the second glucosylation step in the sophorolipidbiosynthetic pathway of Candida bombicola, or a fragment thereof havingsaid UDP-glucosyltransferase activity or a variant thereof having atleast 50% sequence identity with SEQ ID No 4 and having saidUDP-glucosyltransferase activity. The term ‘fragment’ refers to aprotein containing fewer amino acids than the amino acid sequence asdepicted by SEQ ID No 4 and that retains said UDP-glucosyltransferaseactivity. Such fragment can for example omit N- and C-termini of theprotein leaving a shortened protein of 351 amino acids starting at 18and ending at G358 without hereby touching the UDP-glucosyltransferaseactivity. The term “variant” refers to a protein having at least 50%sequence identity, preferably having at least 51-70% sequence identity,more preferably having at least 71-90% sequence identity or mostpreferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99%sequence identity with SEQ ID No 4 or with a fragment thereof, and, thatencodes for a protein retaining said UDP-glucosyltransferase activity.The latter may differ from the protein as depicted by SEQ ID No 4 or afragment thereof only in conservative substitutions and/ormodifications, such that the ability of the protein to haveUDP-glucosyltransferase activity is retained. A “conservativesubstitution” is one in which an amino acid is substituted for anotheramino acid that has similar properties, such that one skilled in the artof protein chemistry would expect the nature of the protein to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be proteins as described hereinmodified by, for example, by the deletion or addition of amino acidsthat have minimal influence on the UDP-glucosyltransferase activity,secondary structure and hydropathic nature of the protein. Regionswithin SEQ ID No 4 which contribute to the proteins' activity areresidues 18 to V48, G129 to C153, I170 to E186, L203 to F223, S261 toY277, V334 to H350 and A371 to T382. Hence, the variants as definedabove preferably comprise at least one of the latter regions. Morepreferably, the latter variants comprise at least one of the followingresidues or amino acids of SEQ ID No 4: G17, H18, G20, R175, V178, F179,G182, R212, T272, N338, G340, G342, G343 or H346.

The present invention further relates to the expression of SEQ ID No 1,SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4 or variants thereof in otherorganisms than the various yeast species capable of producingsophorolipids as specified above. These ‘other’ organisms can bemicro-organisms such as bacteria, yeast and fungi, plants, animals andalgae. Expression of the genes in these organisms leads to theproduction of useful compounds such as glycosylated compounds,glycolipids or sophorolipids.

As specified above, the present invention specifically refers to the useof SEQ ID No 1, SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4 and variantsthereof in yeast species capable of producing sophorolipids. Thesesequences can either be manipulated in the endogenous strain (e.g. in C.bombicola) by knocking out the gene or can be subjected to otheralterations such as mutation or overexpression. These manipulations canbe obtained by various methods known by the person skilled in the art.Alteration of the expression will result in modification of a glycolipidbiosynthetic pathway, more in particular the sophorolipid biosyntheticpathway. The invention has further relates to the usage of a modifiedyeast strain as indicated above for the production of compounds such asrecombinant proteins (e.g. GFP and amylase), beta-hydroxy fatty acidsand polyhydroxyalkanoates (PHA), dicarboxylic acids, polyunsaturatedfatty acids, hydroxylated fatty acids: terminal or subterminalhydroxylated or at any other position, glycolipids such ascellobioselipids, glucolipids, trehaloselipids rhamnolipids,sophorolipids with a special fatty acid tail, sophorolipids with a fattyacid tail ranging from 10 to 15 carbon atoms, sophorolipids with a fattyacid tail of 17 carbon atoms, sophorolipids with a fatty acid tailranging from 19 till 25 carbon atoms, sophorolipids with branched fattyacid tail, sophorolipids with multiple hydroxylated fatty acid tail,fully lactonized sophorolipids and fully acidic sophorolipids; rhamnose,sophorose, polyketide antibiotics, lactonic structures (fatty acidbased), organic acids such as succinate, adipate and citrate;oleagenious compounds, hydrophobic compounds, sophorose, rhamnose,squaleen, vitamin D, resveratrol, steroids, and carotenoides.

The invention further specifically relates to the usage as indicatedabove wherein said compounds are glycolipids (such as cellobioselipidsand glucolipids), polyhydroxyalkanoates or any other oleaginouscompounds and recombinant proteins such as green fluorescent protein andamylase.

The above-indicated usages can be performed using well known techniquesof genetic engineering.

The present invention and the above-indicated usages will be illustratedby the following non-limiting examples.

EXAMPLES Example 1 Cytochrome P450 Monooxygenase CYP52M1 Knock-Out

Introduction

This strain is knocked-out at the CYP52M1 gene (GenBank accession numberEU552419), encoding for the enzyme responsible for the hydroxylation offatty acids with a preferred length of 16 or 18 carbon atoms. The fattyacid is converted to a ω or ω-1 hydroxylated fatty acid. In this strain,hardly any sophorolipid production is detected, while cell growth andviability is comparable to the wild type.

Materials and Methods

Strains, Plasmids and Culture Conditions

Candida bombicola ATCC 22214 was used in all experiments. Whensophorolipid production was intended, the medium described by Lang etal. (Lang et al., 2000) was used. Yeast cultures were incubated at 30°C. and 200 rpm.

All PCR products intended for sequence analysis were cloned into thepGEM-T® vector (Promega). Escherichia coli DH5α was used in all cloningexperiments and was transformed as described by Sambrook and Russell(2001). E. coli cells were grown in Luria-Bertani (LB) medium (1%trypton, 0.5% yeast extract and 0.5% sodium chloride) supplemented with100 mg/L ampicillin and 40 mg/L X-gal if necessary. Liquid E. colicultures were incubated at 37° C. and 200 rpm.

DNA Isolation and Sequencing

Yeast genomic DNA was isolated with the GenElute™ Bacterial Genomic DNAKit (Sigma). Preceding protoplast formation was performed by incubationat 30° C. for 90 minutes with zymolyase (Sigma).

Bacterial plasmid DNA was isolated with the QIAprep Spin Miniprep Kit(Qiagen). All DNA sequences were determined at the VIB Genetic ServiceFacility (Belgium).

Transformation

C. bombicola cells were transformed with the lithium acetate method(Gietz et al., 1995), but 50 mM LiAc was used instead of 100.Transformants were selected on yeast peptone dextrose (YPD) plates (1%yeast extract, 2% peptone, 2% glucose and 2% agar) containing 500 μg/mLhygromycin or on synthetic dextrose (SD) plates [0.67% yeast nitrogenbase without amino acids (DIFCO) and 2% glucose]. E. coli cells weretransformed as described by Sambrook (Sambrook et al., 2001).

Creation of the Knock-Out Cassette

The 1617 bp coding fragment and 218 and 1060 bp upstream and downstreamof the CYP52M1 gene were amplified with the primers A21TotFor andA21TotRev (Table 1), yielding a fragment of 2869 bp which was clonedinto the pGEM-T® vector (Promega). The created vector was digested withAvaI, cutting the coding sequence of CYP52M1 twice, in this way deleting308 bp of the CYP52M1 sequence. The E. coli hygromycin resistance genecontrolled by the Candida bombicola GPD promoter (Van Bogaert et al.,2008a) was inserted by means of the In-Fusion™ 2.0 Dry-Down PCR CloningKit (Clontech). The primers GHInfA21 For and HygroInfA21Rev weredesigned according to the guidelines of the manual and used forintegration of the hygromycin resistance cassette (1968 bp) intoCYP52M1. The primerpair A21KnockHygroCasFor and A21KnockHygroCasRev wereused for the amplification of a 4003 bp fragment containing thehygromycin resistance cassette with approximately 1000 bp of the CYP52M1sequence on each site. The fragments were used to transform the Candidabombicola wild type strain.

Sampling

Analytical sophorolipid samples were prepared as follows: 440 μLethylacetate and 11 μL acetic acid were added to 1 mL culture broth andshaken vigorously for 5 min. After centrifugation at 9 000 g for 5 min,the upper solvent layer was removed and put into a fresh eppendorf tubewith 600 μL ethanol. At the end of the incubation period, 3 volumesethanol were added to the culture broth for total extraction ofsophorolipids. Cell debris was removed by centrifugation at 1500 gduring 10 min. Samples were analysed by HPLC and Evaporative LightScattering Detection.

Cell dry weight (CDW) was measured by centrifugation of 2 mL culturebroth for 5 min at 9 000 g. Pellets were washed two times with ethanolto remove sophorolipids and hydrophobic substrate and finally dissolvedin distilled water. The suspension was transferred to a cellulosenitrate filter with a pore diameter of 0.45 μm (Sartorius) and the dryweight was determined in the XM60 automatic oven from PrecisaInstruments Ltd.

Colony forming units (CFU) were determined by plating decimal dilutionson agar plates with 10% glucose, 1% yeast extract and 0.1% urea whichwere incubated at 30° C. for three days.

HPLC-Analysis of Sophorolipids

Sophorolipid samples were analysed by HPLC on a Varian Prostar HPLCsystem using a Chromolith® Performance RP-18e 100-4.6 mm column fromMerck KGaA at 30° C. and Evaporative Light Scattering Detection(Alltech). A gradient of two eluents, a 0.5% acetic acid aqueoussolution and acetonitrile, had to be used to separate the components.The gradient started at 5% acetonitrile and linearly increased till 95%in 40 min. The mixture was kept this way for 10 min and was then broughtback to 5% acetonitrile in 5 min. A flow rate of 1 mL/min was applied.

Results and Discussion

The CYP52M1 knock-out cassette was constructed as described in theMaterials and Methods section. This linear fragment was used totransform Candida bombicola wild type cells. The genotype of 10transformants was checked by yeast colony PCR with the primerHygroInsertCheckFor, binding on the knock-out cassette and primerA21totRev, binding the genomic CYP52M1 gene (Table 1). All 10transfromants displayed the right genotype. The effect of the disruptedCYP52M1 gene was tested by evaluating three randomly selectedtransformants for their production of sophorolipids in liquid medium.CDW and colony forming unit were similar as compared to the wild typestrain, indicating that the gene disruption did not affectedcell-growth.

However, clear differences were observed regarding glucose consumption.Whereas during the exponentional growth phase no differences wereobserved, the wild type shows much faster consumption in the stationarygrowth phase, when sophorolipid synthesis takes place. As sophorolipidproduction demands a large glucose input, low glucose utilization of theknock-outs suggests the absence of sophorolipid synthesis. Furthermore,during the whole incubation period rapeseed oil staid floating on theculture medium surface of the knock-outs, indicating that also thehydrophobic carbon source required for sophorolipid production is notconsumed.

Finally, biosurfactant production was checked by HPLC analysis ofsamples taken during and at the end of the incubation period. Whereasthere was a clear production for the wild type, no sophorolipids couldbe detected in the medium or the cells of all tree knock-outs (FIG. 1).The peaks observed for the transformant are degradation products of thenot consumed rapeseed oil: oleic acid, the major constituent of rapeseedoil fatty acids (60%) is detected at 36.2 minutes and also linoleic acid(23%) is identified (33.5 min).

The above mentioned results demonstrate that CYP52M1 is the cytochromeP450 monooxygenase responsible for the synthesis of hydroxylated fattyacids, which are essential for sophorolipid production. Disabling of thegene inhibits sophorolipid synthesis, but has surprisingly no effect oncell growth or viability. The created strains are unable to producesophorolipids and can consequently be used as production host. As C.bombicola is not intoxicated by oily or hydrophobic substrates and hasmetabolic pathways to metabolise or convert those, the knock-out straincan be used as a platform strain for the synthesis of other oleaginousproducts such as polyhydroxyalkanotes. Furthermore, the strains can beused to express heterologous P450 enzymes in order to createsophorolipids with a tailor-made fatty acid tail.

Example 2 Identification of the UDP-Glucosyltransferase Gene UGTA1,Responsible for the First Glucosylation Step in the SophorolipidBiosynthetic Pathway of Candida Bombicola ATCC 22214

Introduction

This strain is knocked-out at the UGTA1 gene (GenBank accession numberHM440973), encoding for the enzyme responsible for the firstglucosylation step in the sophorolipid biosynthetic pathway. This enzymetransfers glucose from UDP-glucose to a hydroxylated fatty acidresulting in the production of a glucolipid.

In this strain, no sophorolipid production is detected, while cellgrowth and viability is comparable to the wild type (FIG. 2).

Materials and Methods

Strains, Plasmids and Culture Media

Candida bombicola ATCC 22214 was used for isolation of the UGTA1 geneand C. bombicola

G9 (Van Bogaert et al., 2008b) for creation of the ugtA1 deletionmutant. Escherichia coli DH5α F′ was used for plasmid maintenance. Yeastcells were grown on YPD medium containing 1% yeast extract, 2% peptoneand 2% dextrose, SD medium containing 0.67% yeast nitrogen base withoutamino acids (Difco) and 2% glucose or 3C medium containing 10% glucose,1% yeast extract and 0.1% ureum. Liquid media were incubated at 30° C.and 200 rpm. E. coli was grown on Luria Bertani medium (0.5% Bacto yeastextract, 1% Bacto Trypton, 0.5% NaCl) containing 0.01% ampicillin andincubated at 37° C. and 200 rpm. Plasmids were isolated from E. coliDH5α F′ by means of the MiniPrep Plasmid Isolation kit from Qiagen.

Cloning of the C. Bombicola UGTA1 Gene

Primer Design and Sequence Analysis

Primer design, sequence analysis and strategy design were performed withthe Clone Manager Professional Suite software (Version 8.0). Primerswere ordered at Sigma and all plasmids created were sent for sequencingeither to VIB (Belgium) or AGOWA (Germany). Homology searches wereperformed with the BLAST program (Altschul et al., 1997) againstdatabases available at the NCBI website.

Isolation of Genomic DNA

C. bombicola ATCC22214 was grown overnight on 3C medium. Minor amountsof sophorolipids were removed by extracting 500 μl culture samples withone volume ethylacetate. Yeast cell wall was removed enzymatically byincubating with 200 units Yeast Lytic Enzyme (Sigma) in SCE buffer (1Msorbitol, 0.1M sodium acetate and 60 mM EDTA, pH 7.0) for 90 min at 37°C. in presence of 3.75 μl mercaptoethanol. Genomic DNA was isolated fromthe remaining protoplasts by means of the GenElute™ Bacterial GenomicDNA kit (Sigma).

Genome Walking

For isolation of the UGTA1 gene sequence from C. bombicola ATCC 22214genomic DNA, the BD GenomeWalker™ Universal Kit (BD Biosciences) wasused. Gene specific primers for primary and nested PCR were designedbased on the partial UGTA1 sequence obtained from preliminary genomesequencing data and are given in Table 2. Amplification reactions wereperformed with the Expand Long Template PCR System (Roche) following theprotocol as described in (De Maeseneire et al., 2006). PCR amplificationproducts are purified either from the PCR mix or by gel extractionmaking use of the Qiaquick PCR purification kit (Qiagen) or Qiaexll gelextraction kit (Qiagen) respectively. Purified fragments were cloned inpGEM-T® using pGEM-T® Vector System (Promega) and resulting plasmidsused for transformation of E. coli DH5α F′ according to Sambrook(Sambrook et al., 2001). Correct colonies were grown in liquid LB forsubsequent plasmid isolation and sequencing.

Cloning of the Complete UGTA1 Gene

The complete sequence of the UGTA1 gene was amplified from C. bombicolaATCC 22214 genomic DNA by means of the High Fidelity PCR Master Kit(Roche). The obtained 2566 bp fragment was purified and cloned inpGEM-T® as described before. The resulting plasmid was calledpGugtA1Tot.

Creation of the UGTA1 Knock-Out Cassette

The procedure leading to the knock-out cassette for the C. bombicolaUGTA1 gene is based on a restriction enzyme mediated insertion of theURA3 selectable marker (Van Bogaert et al., 2007) between regions ofhomology to the 5′ and 3′ ends of the UGTA1 gene. All PCR reactions wereperformed with the Roche High Fidelity System. In a first step, the 5′(A1P) and 3′ (A1T) regions of the UGTA1 gene were amplified from plasmidpGugtA1Tot by means of restriction primers A1P RevNheI and A1T ForNheIin combination with UDPGTA1 TotF and A1T Rev to amplify UGTA1 5′ (A1P)and UGTA1 3′ (A1T) regions respectively (Table 2). These fragments of853 bp and 1121 bp respectively were purified and digested with NheIrestriction enzyme (New England Biosystems) according to Sambrook(Sambrook et al., 2001). The digested fragments were purified from therestriction mixes and ligated by means of the T4 DNA ligase (Fermentas).The ligation product was amplified again and purified by gel extraction.Subsequently this fragment (A1PT) was cloned in pGEM-T®, plasmid pGA1PTwas obtained from E. coli and checked by sequencing as describedearlier. Since pGEM-T® lacks a NheI restriction site, the obtainedplasmid pGA1PT can be linearised solely at the introduced NheI sitebetween A1P and A1T fragments, giving rise to sticky ends at both sites.Accordingly, the URA3 marker is amplified from plasmid pCbura3 (VanBogaert et al., 2007) making use of restriction primers Ura3 FbisNheIand Ura3 RbisNheI that both contain NheI restriction site extensions attheir 5′ends. After purification, the obtained fragment was digestedwith NheI and ligated into the linearised pGA1 PT vector by means of theT4 DNA ligase (Fermentas). The ligation mixture was used fortransformation of E. coli DH5α and right transformants were grown in LBfor subsequent plasmid isolation. The integration of the URA3 marker wasverified by control digestion with restriction enzyme Accl (New EnglandBiolabs). The complete URA3 gene sequence on the obtained plasmidpGKO_A1 was confirmed by sequencing.

Creation of a ugtA1 Deletion Mutant

A linear knock-out cassette was produced from plasmid pGKO_A1 by highfidelity PCR making use of the outer primers UDPGTA1 TotF and A1T Revsince it has been shown that linearisation increases to a large extentthe recombination frequency (Van Bogaert et al., 2008b). The obtainedfragment was purified and 2.5 μg was used for transformation of theura3⁻ C. bombicola G9. The protocol as described for Saccharomyces wasused (Gietz et al., 1995) with some slight modifications. A 50 mM LiAcsolution was used instead of 100 mM, incubation of cells with thecassette before heat shocking occurred for 90 minutes instead of 30 andno DMSO was added. After transformation, cells were plated on SD agarmedium and incubated at 30° C. until transformant colonies appeared.

Characterisation of the ugtA1 Deletion Mutant

Correct integration of the cassette into the genome was checked by meansof yeast colony PCR using primers that anneal outside the recombinationsites. Transformants with the right genotype were then transferred to 3Cagar plates and tested for sophorolipid production. For that, the mutantyeast colony was inoculated in liquid medium as described by Lang (Langet al., 2000) and grown for 48 hours before addition of rapeseed oil (30g/L). Wildtype Candida bombicola ATCC 22214 was grown the same way andserved as a reference. Ten days after addition of the hydrophobic carbonsource, sophorolipid production was verified by extracting 1 ml culturemedium with 400 μl technical ethylacetate in presence of 10 μl aceticacid. After vortexing for 5 minutes, 300 μl of the solvent phase wasdiluted in 1.7 ml absolute ethanol and analysed on HPLC-ELSD using aVarian ProStar HPLC (Varian) equipped with a Chromolith® PerformanceRP-18e column [100 mm (I)×4.6 mm (I.D.)] (Merck) and connected to anEvaporative Light Scattering Detector (Alltech). Compounds were elutedby means of an acetonitrile/acetic acid (0.5% in water) gradient (5/95to 95/5 in 40 min) under constant flow of 1 ml/min. Column temperaturewas set at 30° C.

Biocatalytic Function of the UgtA1 Glucosyltransferase

Preparation of Cell Lysates

Wildtype yeast and the ugtA1 deletion mutant A113 were inoculated from3C agar medium into 5 ml Lang medium and grown overnight at 30° C. and200 rpm. With this preculture, 50 ml of fresh Lang medium was inoculatedwith a start OD of 0.2 and incubated the same way until cells werelysed. Cells were harvested by centrifugation at 3000 rpm and 4° C. witha swinging bucket centrifuge and washed with 10 ml distilled water. Thepellet was then resuspended in lysis buffer pH 7.7 containing 50 mMKH₂PO₄, 5% glycerol, 0.5 mM MgCl₂, 0.5 mM DTT and 1 mM PMSF to OD₁₀₀. Anequal volume of acid washed glass beads (150-212 μm diameter, Sigma) wasadded and cells were disrupted by vortexing during 15 minutes with 30seconds intervals on ice. Soluble protein fractions were used for enzymeassays after centrifugation of the crude lysate at 3000 rpm at 4° C.Protein concentration in the lysate was determined by means of the BCA™Protein Assay Kit (Pierce). Protein solutions were stored at 4° C.

Enzyme Assays

UDP-glucose was obtained from Sigma, 17-hydroxyl-octadecenoic acid andglucolipid were obtained from sophorolipids as described before (Saerenset al., 2009). All substrate solutions were prepared freshly in 50 mMKH₂PO₄, pH 7.7. Enzyme assays contained 2 mM UDP-glucose, 2 mM acceptorand 200 μl fresh protein solution in a total volume of 250 μl. For theblank reactions phosphate buffer replaced either the donor, the acceptoror the protein solution. For each assay also a blank reaction with 200μl buffer was performed. All enzyme reactions were incubated at 30° C.for 3 hours. Reactions were stopped by addition of 200 μl HCl (2N) andglycolipids were extracted with 800 μl diethylether/ethylacetate (1/1).From the solvent phase, 700 μl was recovered, evaporated to dryness andredissolved in 300 μl absolute ethanol before analysis on HPLC asdescribed above.

Results and Discussion

Isolation of the UGTA1 Gene Sequence

Preliminary genome sequencing data yielded part of a putativeglucosyltransferase with homology to glycosyltransferases of the MGTfamily and other UDP-glucuronosyl/UDP-glucosyltransferases available atthe NCBI databases. The alignments illustrated that this partial ORFcontained an appropriate start codon but lacked a stop codon. In orderto isolate the complete ORF, primers were designed for further genomewalking downstream of this putative gene. In this way, another 1600 bpcould be obtained that after assembling with the primary sequenceresulted in a total raw sequence fragment of 2566 bp. This fragmentcontained a complete open reading frame of 1392 bp encoding a putativeUDP-glucuronosyl/UDP-glucosyltransferase gene of 463 amino acids whichwe named UgtA1.

Cloning and Sequence Analysis of the Complete UGTA1 Gene

To isolate the complete UGTA1 gene from the Candida bombicola genome,the 2566 bp fragment was amplified by high fidelity PCR on freshlyisolated genomic DNA. The encoding region with 233 bp and 153 bp of theup- and downstream regions respectively is given in FIG. 3. The openreading frame encodes a putative glycosyltransferase of 463 amino acidswith an estimated molecular mass of 50.5 kDa and estimated pl of 5.45.

Though most yeast promoters lack a clear TATA box, an NT rich TATA-likeconsensus sequence TATA(A/T)A(A/T)(A/G) could be identified 50-200 bpupstream of highly regulated or stress-induced genes from Saccharomyces(Basehoar et al., 2004). If comparable regulatory mechanisms would existfor the transcriptional activation of housekeeping and stress-inducedgenes in C. bombicola, and since sophorolipid production is linked tonitrogen limiting conditions (Davila et al., 1992) one might expect acomparable TATA-like consensus in genes involved in sophorolipidproduction by this yeast. One possible promoter element with highhomology to the Saccharomyces consensus could be identified 415 bpupstream of the UGTA1 startcodon (results not shown), while a second A/Trich region is found closer to the startcodon (FIG. 3). Also severalpossible polyadenylation sequences are found but no specific signalelements involved in 3′-end formation of mRNA like described for yeast(Guo et al., 1996) can be distinguished.

Another typical feature for genes regulated by nitrogen metabolism isthe presence of GATA-like regulatory sequences in their upstreamregions. Such GATA motifs are recognized by GATA-type transcriptionfactors which are strongly activated by depletion of nitrogen (Magasaniket al., 2002). One typical example of such GATA-type transcriptionfactor is the AreA gene of Aspergillus nidulans (Marzluf et al., 1997)and several GATA-like elements are recognized in glycolipid synthesizinggenes of Ustilago maydis [Hewald et al., 2005]. In the 5′ upstreamregion of the UGTA1 encoding region several possible GATA-like elementsare found.

Sequence Homology to Other UDP-Glucosyltransferases

Analysis of the UgtA1 protein sequence via the Conserved Domain Databaseavailable at the NCBI website (Marchler-Bauer et al., 2009), shows thatthis protein belongs to the GT1 family of glycosyltransferases (E.C.2.4.1), a polyspecific family that harbours to date over 3200 differentglycosyltransferases of which most are inverting enzymes usingnucleotide-activated sugars or sugar phosphates as donor molecules(Campbell et al., 1997, Coutinho et al., 2003). The structures of theGT1 proteins show the typical GT-B type topology, which is reflected ina conserved domain (cl 10013) that is also found in the UgtA1 sequenceand therefore this family is taken up in the GT-B type superfamily ofglycosyltransferases (Teichmann et al., 2007). Otherglycosyltransferases such as the RhIB from P. aeruginosa (Ochsner etal., 1994a) and both the Emt1 and Hgt1/Ugt1 proteins from U. maydis(Hewald et al., 2005, Teichmann et al., 2007), which catalyze analogousglycosylation reactions in the glycolipid biosynthetic pathways of theseorganisms, belong to the same GT1 family. Surprisingly, the overallUgtA1 protein sequence shows only low similarity to these proteins (34%similarity to the Ustilago Hgt1/Ugt1 and 38% similarity to the P.aeruginosa RhIB) as derived from a global pairwise alignment usingBioEdit Software (Hall, 1999). Moreover, a Blastp homology searchillustrates that the UgtA1 protein shows poor similarity to any otherprotein sequence. The highest homology is found with bacterialUDP-glucose/UDP-glucuronosyltransferases of which most belong to the MGTsubfamily (Table 3), though the sequence identity is still low. Proteinsof the MGT subfamily are involved in biosynthesis or inactivation ofmacrolide antibiotics and show a conserved domain (TIGR01426/cd 03784)which is also found in the UgtA1 sequence, indicating the transfer of asugar moiety from a nucleotide activated sugar residue to a complexacceptor such as the heptapeptide core of an antibiotic. These findingsindicate that the UgtA1 protein is very likely involved in the synthesisof a complex metabolite.

Creation and Characterization of a ugtA1 Deletion Mutant

The knock-out cassette used is a linear fragment based on a restrictionenzyme mediated insertion of the URA3 selectable marker between 853 bpand 1024 bp of homology to the 5′ and 3′ regions of the UGTA1 generespectively. This cassette is used for transformation of the ura3deficient strain G9. Only when homologous recombination has occurred andthe cassette is integrated in the genome, URA3 functionality will berecovered and transformants will be able to grow on SD medium, lackinguracil. Five days after transformation of the G9 strain, 40 colonieswere obtained on the selective plates, of which 17 were subjected to ayeast colony PCR to check their genotype. Only one colony showed theright genotype and is referred to as C. bombicola A113. For the other 16selected transformants, ura3 complementation will have occurred eitherby recombination between the marker only and the ura3 mutant allele ofthe G9 strain or by one sided or illegitimate recombination.

To control the involvement of the UgtA1 glucosyltransferase insophorolipid production, the obtained mutant C. bombicola A113 was grownin production medium as described before and sophorolipid production wascompared to the wildtype yeast. FIG. 4 shows the HPLC chromatograms ofculture extracts from both C. bombicola A113 and wild type yeast whenrapeseed oil was used as hydrophobic carbon source. Only the wild typeyeast strain produces sophorolipids while the ugtA1 deletion mutant grewwell but completely lost its ability to produce any glycolipid. Thisclearly indicates that the UgtA1 glucosyltransferase has a key-functionin sophorolipid production by the yeast. Since the mutant shows acomparable growth as the wildtype yeast, it is unlikely that the UgtA1glucosyltransferase has any other function in primary metabolism.

Biocatalytic Function of the UgtA1 Glucosyltransferase

In order to link the UgtA1 glucosyltransferase to a specific step in thesophorolipid biosynthetic pathway, glucosyltransferase activities weremeasured in cell lysates of the A113 mutant and compared to that of thewildtype yeast. Since sophorolipid production is linked to nitrogenstarvation conditions, it is very likely that the expression of genesinvolved in the biosynthetic pathway only start in early stationaryphase. To determine the best time point to harvest cells for proteinextraction and subsequent activity tests, wildtype C. bombicola wasgrown in Lang medium and glucosyltransferase activities were determinedin 5 ml culture samples taken at different time points. For detection ofthe first glucosyltransferase activity (GTI), UDP-glucose and17-hydroxyoctadecenoic acid were used as glucosyl donor and acceptorrespectively. For detection of the second activity (GTII), the fattyacid was replaced by the glucolipid. In the wildtype yeast bothglucosyltransferase activities were detected from late exponential phaseand further increase to a maximum in early stationary phase. Addition ofrapeseed oil to the culture medium after 48 hours did not result inincreased activities while no significant activity could be detectedwhen cells were grown in standard yeast media such as YPD (results notshown). Therefore the best set up for the enzyme assays is to harvestcells after 42 to 48 hours of incubation in Lang medium without additionof hydrophobic carbon source. Under these conditions, lysates wereprepared from both wildtype yeast and the A113 mutant and solubleprotein fractions were used for the same glucosyltransferase assays. Theresults are presented in FIGS. 5 and 6. From FIG. 5 it is clear thatboth GTI and GTII activities are present in the cell lysate from thewildtype yeast, since both glucolipids (GTI activity) and sophorolipids(GTII activity) are detected as products. None of these activities wereobserved when either cell lysate, UDP-glucose or fatty acid was omittedfrom the reaction. In contrast to the wildtype lysate, the cell lysateof the A113 mutant does not show any glucosylation product from thefatty acid what indicates that the UgtA1 protein is at least responsiblefor the first glucosylation reaction in the sophorolipid biosyntheticpathway. The signals at 27 and 29 minutes in the chromatograms of thewildtype assays, come from de novo synthesis of sophorolipids duringgrowth in Lang medium. These peaks are absent in the chromatograms ofthe A113 mutant, confirming that de novo synthesis in this mutant iscompletely blocked. In order to verify if the second glucosylation step(GTII) is affected by the disruption of the UgtA1 protein, the assay wasrepeated with glucolipid as the acceptor. From FIG. 6 it can be clearlyseen that the second glucosylation activity is still present in the A113lysate and that this activity is comparable to that of the wildtypelysate. We thus provide evidence that sophorolipid production in C.bombicola involves two different glucosyltransferases that transferglucose from UDP-glucose to their respective acceptor substrates in astepwise but independent manner. We show that the UgtA1glucosyltransferase isolated here, is responsible for the firstglucosylation reaction and disrupting the gene has no influence on thesecond glucosylation step. This second glucosyltransferase appears to behighly specific towards its own acceptor substrate, since no glucolipidformation is observed when fatty acids are present as acceptor in theA113 assays (FIG. 5). Based on these findings, we believe thatsophorolipid production in C. bogoriensis relies on a comparablestepwise pathway and that it is doubtful that a multifunctional proteinwould exist accepting both fatty acid and glucolipid in its catalyticcentre.

Conclusion

Here we identified the gene UGTA1 with a clear function in sophorolipidproduction by Candida bombicola. The UGTA1 gene encodes aglucosyltransferase of 463 amino acids and an estimated molecular weightof 50.5 kDa. The protein can be classified within the GT1 family ofglycosyltransferases (EC 2.4.1.x). By the creation of a ugtA1 deletionmutant we could identify that this protein has a clear function insophorolipid production and enzyme assays on cell lysates providedfurther evidence that the UgtA1 glucosyltransferase is catalyzing thefirst glucosylation step in the sophorolipid biosynthetic pathway of C.bombicola. We further demonstrated that the second glucosylationreaction is catalyzed by another independent glucosyltransferase andthat this transferase is highly specific towards glucolipids as asubstrate, since disrupting the UgtA1 activity results in a completeloss of sophorolipid production both in vivo and in vitro. The A113strain created here is thus no longer capable to convert ω or ω-1hydroxylated fatty acids to glucolipids and is therefore useful inproduction of hydroxy fatty acid based compounds. The lack of GTIactivity will lead to an intracellular pool of hydroxylated fatty acidswhich can be used as building blocks for other oleaginous compounds.

Example 3 Identification of the UDP-Glucosyltransferase Gene UGTB1Responsible for the Second Glucosylation Step in the SophorolipidBiosynthetic Pathway of Candida Bombicola ATCC 22214

Introduction

This strain is knocked-out at the UGTB1 gene (GenBank accession numberHM440974), encoding for the enzyme responsible for the secondglucosylation step in the sophorolipid biosynthetic pathway. The enzymetransfers glucose from UDP-glucose to a glucolipid, resulting in theproduction of a sophorolipid.

In this strain, no sophorolipid production is detected, while cellgrowth and viability is comparable to the wild type (FIG. 2).

Materials and Methods

Strains, Plasmids and Culture Media

Candida bombicola ATCC 22214 was used for isolation of the UGTB1 geneand C. bombicola G9 (derived from ATCC 22214, see Van Bogaert et al.,2008b) for creation of the ΔugtB1 deletion mutant. Escherichia coli DH5αF′ was used for plasmid maintenance. Yeast cells were grown on YPDmedium containing 1% yeast extract, 2% peptone and 2% dextrose, SDmedium containing 0.67% yeast nitrogen base without amino acids (Difco)and 2% glucose or 3C medium containing 10% glucose, 1% yeast extract and0.1% urea. Liquid media were incubated at 30° C. and 200 rpm. E. coliwas grown on Luria Bertani medium (0.5% Bacto yeast extract, 1% BactoTrypton, 0.5% NaCl) containing 0.01% ampicillin and incubated at 37° C.and 200 rpm.

Plasmids were isolated from E. coli DH5α F′ by means of the MiniPrepPlasmid Isolation kit from Qiagen and sequenced at AGOWA (Germany).

Primer Design and Sequence Analysis

Primer design, sequence analysis and strategy design were performed withthe Clone Manager Professional Suite software (Version 8.0). Primerswere ordered at Sigma.

Cloning of the Complete UG TB 1 Gene

For isolation of genomic DNA, C. bombicola ATCC22214 was grown overnighton 3C medium. Minor amounts of sophorolipids were removed by extracting500 μl culture samples with one volume ethylacetate. Cell wall wasremoved enzymatically by incubating with 200 units Yeast Lytic Enzyme(Sigma) in SCE buffer (1M sorbitol, 0.1M sodium acetate and 60 mM EDTA,pH 7.0) for 90 min at 37° C. in presence of 0.75% β-mercaptoethanol.Genomic DNA was isolated from the remaining protoplasts by means of theGenElute™ Bacterial Genomic DNA kit (Sigma).

The complete sequence of the UGTB1 gene was amplified from C. bombicolaATCC 22214 genomic DNA by means of the High Fidelity PCR Master Kit(Roche) and the primers GTII−472For and GTII+239Rev (Table 4). Theobtained fragment was purified by means of the Qiaquick PCR purificationkit (Qiagen) and cloned in pGEM-T® making use of the pGEM-T® VectorSystem (Promega). This led to the plasmid pGugtB1 Tot which was thenused for transformation of E. coli DH5α F′ according to (Sambrook andRussell, 2001). Correct transformants were isolated after colony PCR andgrown in liquid LB for subsequent plasmid isolation.

Creation of the UGTB1 Knock-Out Cassette

The knock-out cassette for the C. bombicola UGTB1 gene is based on theintegration of the URA3 selectable marker between regions of homology tothe 5′ and 3′ termini of the UGTB1 gene. In a first step, plasmidpGugtB1 Tot is linearized by a double digest with single cutting enzymesAvaI and KasI (New England Biolabs) according to Sambrook (Sambrook andRussell, 2001). In this way a fragment of 100 bp (from by 950-1049 ofthe insert) is removed from the UgtB1 encoding sequence, leaving stickyfragments at both ends of the linearized vector. The URA3 selectionmarker was obtained after high fidelity PCR with the PfuUltra HighFidelity PCR system (Stratagene) on plasmid pCbura3 (Van Bogaert et al.,2008). Primers used were ura3infugtB1 F and ura3infugtB1 R (Table 4).These primers contain 15 bp of homology to respectively the 3′ and 5′end of the linearized plasmid pGugtB1 Tot. The obtained amplicon wasused directly for cloning in the linearized plasmid making use of theIn-Fusion Dry Down PCR cloning kit (Clontech). This led to the plasmidpGKO_ugtB1 which now contains the complete C. bombicola ATCC22214 URA3gene (2043 bp) flanked by 949 bp and 961 bp of homologous regions to theUGTB1 5′ and 3′ sequence respectively. Cloning and subsequenttransformation of Fusion Blue competent E. coli cells, for maintenanceof the plasmid, were done as described in the manual of the kit. E. colitransformants were tested by colony PCR for insertion of the URA3 markerin the plasmid and subsequently grown in LB for plasmid isolation. Thegene sequence of the URA3 gene was confirmed by sequencing.

Creation of a ΔugtB1 Deletion Mutant

A linear knock-out cassette was produced from plasmid pGKO_B1 byPfuUltra High Fidelity PCR (Stratagene) making use of the primersGTII−472F and GTII+239R (Table 4) since recombination frequency isincreased strongly by using linear fragments (Van Bogaert et al., 2008).The obtained fragment was column purified and 1 μg was used fortransformation of the ura3⁻ C. bombicola G9.

For transformation of C. bombicola G9 the protocol as described forSaccharomyces was used (Gietz and Schiestl, 1995) with some slightmodifications. A 50 mM LiAc solution was used instead of 100 mM,incubation of cells with the cassette before heat shocking occurred for90 minutes instead of 30 and no DMSO was added. After transformation,cells were plated on SD agar medium and incubated at 30° C. untiltransformant colonies appeared.

Characterisation of the ΔugtB1 Deletion Mutant

The genotype of the obtained mutants was controlled by yeast colony PCRusing the primers KOugtB1 CtrlF and KOugtB1 CtrlR (Table 4) annealingupstream the left-sided recombination site and the URA3 markerrespectively. To study the phenotype, mutant yeast colonies were grownin liquid medium as described by Lang et al. (2000) for 48 hours beforeaddition of rapeseed oil (37.5 g/L). Wildtype Candida bombicola ATCC22214 served as a reference.

Seven days after addition of the hydrophobic carbon source, sophorolipidproduction was verified by extracting 1 ml culture medium with 400 μltechnical ethylacetate in presence of 10 μl acetic acid.

After vortexing for 5 minutes, 300 μl of the solvent phase was dilutedin 1.7 ml absolute ethanol and analyzed on HPLC-ELSD using a VarianProStar HPLC (Varian) equipped with a Chromolith® Performance RP-18ecolumn [100 mm (I)×4.6 mm (I.D.)] (Merck) and connected to anEvaporative Light Scattering Detector (Alltech). Compounds were elutedby means of an acetonitril/acetic acid (0.5% in water) gradient (5/95 to95/5 in 40 min) under constant flow of 1 ml/min. Column temperature wasset at 30° C. To check the molecular masses of the produced glucolipids,the same samples were analysed under the same conditions on a ShimadzuLC-10-AD HPLC system connected to a quadrupole mass spectrometer(Waters). Molecules were identified by their native molecular massesafter ESI (electron spray ionisation) without collision.

Biocatalytic Function of the UgtB1 Glucosyltransferase

C. bombicola ATCC 22214 and the ΔugtB1 deletion mutant B11 wereinoculated from 3C agar medium into 5 ml Lang medium and grown overnightat 30° C. and 200 rpm. With this preculture, 50 ml of fresh Lang mediumwas inoculated with a start OD of 0.2 and incubated the same way during60 hours. Cells were harvested by centrifugation at 5600 g and 4° C.with a swinging bucket centrifuge and washed with 10 ml distilled water.The pellet was then resuspended in lysis buffer pH 7.7 containing 50 mMKH₂PO₄, 5% glycerol, 0.5 mM MgCl₂, 0.5 mM DTT and 1 mM PMSF to OD₁₀₀. Anequal volume of acid washed glass beads (150-212 μm diameter, Sigma) wasadded and cells were disrupted by vortexing during 15 minutes with 30seconds intervals on ice. Soluble protein fractions were used for enzymeassays after centrifugation of the crude lysate at 5600 g at 4° C.Protein concentration in the lysate was determined by means of the BCA™Protein Assay Kit (Pierce).

UDP-glucose was obtained from Sigma, 17-hydroxy-octadecenoic acid andglucolipid were obtained from sophorolipids as described before (Saerenset al., 2009). All substrate solutions were prepared freshly in 50 mMKH₂PO₄, pH 7.7. Enzyme assays contained 2 mM UDP-glucose, 2 mM acceptorand 200 μl fresh protein solution in a total volume of 250 μl. For theblank reactions, buffer replaced either UDP-glucose, acceptor or proteinsolution. All enzyme reactions were incubated at 30° C. for 3 hours.Reactions were stopped by addition of 200 μl HCl (2N) and glycolipidswere extracted with 800 μl diethylether/ethylacetate (1/1) according toBreithaupt and Light (1982). From the solvent phase, 700 μl wasrecovered, evaporated to dryness and redissolved in 300 μl absoluteethanol before analysis on HPLC as described above. Peak areas wereconverted to product concentrations and enzyme activity was expressed inμM/mg min.

Results and Discussion

Cloning and Sequence Analysis of the Complete UGTB1 Gene

Preliminary genome sequencing data of Candida bombicola ATCC 22214revealed a putative open reading frame (ORF) of 1299 bp with homology toa huge number of mostly hypotheticalUDP-glycosyltransferases/glucuronosyltransferases of microbial origin asshown by a BLASTx homology search (Altschul et al., 1997). Primers weredesigned 472 bp up- and 239 bp downstream of respectively start- andstopcodon of the putative gene and the complete gene sequence wasisolated and cloned into the plasmid pGugtB1 Tot. The gene, referred toas UGTB1 (FIG. 7), encodes a putative protein of 432 amino acids with anestimated molecular mass of 46.2 kDa and estimated pl of 4.98.

Though most Saccharomyces promoters lack a clear TATA box, an NT richTATA-like consensus sequence TATA(A/T)A(A/T)(A/G) could be identified50-200 bp upstream of highly regulated or stress-induced genes (Basehoaret al., 2004). If comparable different regulatory mechanisms would existfor the transcriptional activation of housekeeping and (stress-) inducedgenes in C. bombicola, one might expect a comparable TATA-like consensusin genes involved in sophorolipid production by this yeast as well,since sophorolipid synthesis is observed under nitrogen limitingconditions (Davila et al., 1992). A clear TATA-like elementcorresponding to the Saccharomyces consensus sequence can be identified50 bp upstream the UGTB1 startcodon (FIG. 7). In contrast, no clearpolyadenylation signal or any other signal element that might beinvolved in 3′-end formation of yeast mRNA can be found (Guo andSherman, 1996). Since sophorolipid synthesis is strongly linked tonitrogen limitation, one might expect GATA-like regulatory sequences inthe upstream regions of genes involved in this pathway. Such GATA motifsare recognized by GATA-type transcription factors which are stronglyactivated by depletion of nitrogen (Magasanik and Kaiser, 2002). Onetypical example of such GATA-type transcription factor is the AreA geneof Aspergillus nidulans (Marzluf et al., 1997) and several GATA-likeelements are recognized in glycolipid synthesizing genes of Ustilagomaydis (Hewald et al., 2005). In the 5′ upstream region of the UGTB1gene two possible GATA-like elements can be found (FIG. 7). Takentogether, it is very likely that expression of the isolated UGTB1 geneis under control of a regulation system linked to nitrogen metabolism.

Sequence Homology to Other UDP-Glucosyltransferases

Analysis of the UgtB1 protein sequence against the Conserved DomainDatabase (CDD) (Marchler-Bauer et al., 2009) illustrates that theprotein belongs to the polyspecific GT1 family of glycosyltransferases(Campbell et al., 1997; Coutinho et al., 2003) which is characterized bya GT1_Gtf_like conserved domain (CDD/cd03784) and which includes amongstothers a group of homologous glycosyltransferases involved in the finalstages of vancomycin and chloroeremomycin biosynthesis. These proteinstransfer sugar moieties from an activated NDP-sugar donor to theheptapeptide core of the antibiotic. All 14 conserved catalytic aminoacids are found in the UgtB1 sequence (FIG. 8). From the alignment withother GT1_Gtf_like proteins, residue H18 is supposed to correspond tothe only one conserved amino acid of the acceptor substrate bindingpocket while residues G17, T272, N338, G340, G342 and G343 are supposedto make up the 6 conserved amino acids from the UDP binding site of thedomain.

Because members of the GT1 family all show the GT-B type topology, thisfamily is taken up into the broad GTB-type superfamily ofglycosyltransferases (Breton et al., 2006), characterized by anotherconserved domain (CDD/cl10013) present in the UgtB1 sequence. Sequencehomology to antibiotic-related glycosyltransferases is confirmed by therecognition of a MGT (macrolide glycosyltransferase) conservedmultidomain (CDD/TIGR01426) and the hits obtained from a BLASTp homologysearch against all non-redundant protein sequences available at the NCBIdatabases. Numerous hits with moderate homology appear to behypothetical glycosyltransferases from both bacterial and fungal originarising from the huge number of genome sequencing projects. The highestsequence homology of 57% (38% sequence identity) is found for ahypothetical protein from the plant pathogenic fungus Sclerotiniasclerotiorum 1980 (gb/EDN94128). While no biochemical function can beascribed to most of the matching proteins, a limited number isassociated with secondary metabolite production and more specifically tothe synthesis of antibiotics (Table 5). These findings suggest that theUgtB1 protein is very likely involved in the biosynthesis of a complexsecondary metabolite.

Since rhamnosyltransferase RhIB (gb/L28170) from Pseudomonas aeruginosa(Ochsner et al., 1994) and both erythritol β-mannosyltransferase Emt1(gb/XP_(—)400732) and hydroxypalmitate glucosyltransferase Hgt1(gb/EAK87174) from Ustilago maydis (Hewald et al., 2005; Teichmann etal., 2007) catalyze comparable biochemical steps during the synthesis ofother glycolipids and because these proteins (all E.C. 2.4.1.-) areclassified within the same GT1 family of glycosyltransferases, it wasexpected that UgtB1 would show significant sequence homology to those.Surprisingly, an optimal global pairwise alignment using BioEditSoftware (matrix blosum 62) (Hall et al., 1999) shows 36% sequencesimilarity (22% identity) to RhIB, 33.5% (18%) to Emt1 and 33% (19%) toHgt1, values which are even lower as compared to those for antibioticsynthesizing proteins from bacterial origin (Table 5).

With these characteristics, UgtB1 seems to be very comparable to anotherUDP-glucosyltransferase referred to as UgtA1 (gb/HM440973) we isolatedfrom C. bombicola and to which the first glucosylation step insophorolipid biosynthesis could be ascribed (results not shown).Therefore, sequence homology between these two proteins was verified bymeans of another optimal global pairwise alignment (matrix blosum 62).Both proteins showed 45.2% sequence identity and 61% sequence similarity(FIG. 8). Apart from the GT1_Gtf_like conserved residues, which are alsopresent in the UgtA1 sequence, several other amino acids areconstitutive. Structure homology models that we created for UgtA1 andUgtB1 suggest that these residues are likely situated either in theneighborhood of the catalytic centre or on outer loops and so suggesttheir involvement in substrate recognition and orientation.

Creation and Characterization of a ΔugtB1 Deletion Mutant

A linear knock-out cassette was used for transformation of the ura3⁻Candida bombicola G9, derived from the wildtype yeast ATCC22214 (VanBogaert et al., 2008), and transformants were selected bycomplementation as a result of disruption of the UGTB1 gene by insertionof the URA3 marker. A couple of days after transformation, severaltransformants were obtained on the selective plates and 28 weresubjected to a colony PCR to check their genotype. As a consequence ofprimer annealing sites, either double cross-over events or left-sidedsingle cross over events will lead to an amplicon. From the 28transformants, 13 yielded the expected amplicon. The other mutantsprobably did arise from right-sided single cross-over events,illegitimate recombination or from recombination of the URA3 marker withthe G9 ura3 allele.

Three correct transformants (B11, B14 and B19) were subsequently grownin liquid Lang medium to investigate the influence of the knock-out onsophorolipid production. Wildtype C. bombicola ATCC 22214 was used as areference. The growth of the transformants appeared to be comparable tothe wildtype yeast indicating that the UGTB1 gene has no contribution toany pathway in primary metabolism. Seven days after addition of rapeseedoil, production of sophorolipids in the culture media was checked. FIG.9 shows the results for the wildtype yeast and the B11 mutant (B14 andB19 gave identical results).

In contrast to the wildtype yeast, no sophorolipids were produced by theΔugtB1 deletion mutants indicating that UgtB1 has a key-function insophorolipid production. Instead, the mutants produced glucolipids,indicating that the first glucosylation is still being performed. ThatUgtB1 is catalyzing the second glucosylation was confirmed by enzymeassays on cell lysates of the wildtype yeast ATCC 22214 and the randomlyselected AugtB1 deletion mutant B11. For detection of theglucosyltransferase I activity (GTI), UDP-glucose and17-hydroxy-octadecenoic acid were used as donor and acceptorrespectively. For detection of the glucosyltransferase II activity(GTII), fatty acid was replaced by glucolipid as acceptor. FIG. 10 showsthe analyses of sample extracts after a GTI assay on the lysates ofwildtype and B11 respectively. In the cell lysate of the wildtype,conversion of the fatty acid to glucolipids by the action of the firstglucosyltransferase is followed by a second glucosylation leading tosophorolipids. The peaks at 27 and 29 minutes correspond to diacetylatedlactonised sophorolipids from de novo synthesis during incubation inLang medium. These de novo sophorolipids are absent in the chromatogramof the B11 mutant confirming disruption of the pathway. Since noformation of acidic sophorolipids is observed with the B11 lysate,showing minor formation of glucolipids only, one can conclude that thesecond glucosylation is blocked in the deletion mutant. This isconfirmed by a GTII assay on the same lysates (FIG. 11): whereglucolipids are converted to acidic sophorolipids with the lysate of thewildtype, no conversion of glucolipids is observed with the lysate ofthe B11 mutant. The small peak of fatty acids observed in thesechromatograms originates from glucolipid preparation (Saerens et al.,2009).

Conclusion

Knowledge of the genetic mechanisms behind biosurfactant production bydifferent microorganisms is increasing, opening a new way to increaseyields by creation of overproducing mutants and in that way make thebiosurfactants economically more competitive. The genetics behindsophorolipid production, one of the most promising group of glycolipidbiosurfactants, however remains unclear. Here we isolate a gene from theindustrially applied C. bombicola with a key-function in this importantpathway. The UGTB1 gene encodes the UDP-glucosyltransferase responsiblefor the second glucosylation step as demonstrated by the creation of aAugtB1 deletion mutant and subsequent in vitro enzyme assays with celllysates. We demonstrate here that two independentUDP-glucosyltransferases referred to as UgtA1 and UgtB1 act in astepwise manner. The presence of conserved domains in the UgtB1 proteinsequence indicate that the protein belongs to the GT1 family ofglycosyltransferases. Surprisingly, sequence homology to bacterialglycosyltransferases involved in antibiotic synthesis is higher than thehomology to other glycosyltransferases known so far to be involved inmicrobial glycolipid biosynthesis. This might be explained by thephylogenetic distance between the different organisms. Ustilago maydisfor example is a dimorphic Basidiomycete and the clustering ofglycolipid synthesizing genes in Ustilago suggests that these havearisen from a horizontal gene transfer (Hewald et al., 2006). On thewhole however, sequence homology between the UgtB1 and otherglycosyltransferases of the GT1 family is low which indicates that theUgtB1 might be considered as a new enzyme within this family.

Example 4 Examples of Produced Components Example 4.1 HeterologousProtein Expression

A strain derived from Candida bombicola ATCC 22214, i.e. a strainknocked-out in the cyp52M1 gene (GenBank accession number EU552419), wasused for protein production. In this strain, hardly any sophorolipidproduction is detected, while cell growth and viability is comparable tothe wild type (see example 1).

In order to allow intregration of the heterologous protein gene in thegenome and selection for this event, the ura3-negative PT36 strain wasused. This strains is derived from the wild type strain Candidabombicola ATCC 22214, but only harbors the promotor and terminator ofthe ura3 gene, while the ura3 coding sequence is removed. This was doneby homologous recombination with a cassette containing the ura3 5′upstream non-coding region fused to its 3′ noncoding downstream region.The PT36 mutant are auxotrophic for uracil or uridine (ura3⁻) and can betransformed back to prototrophy with a functional ura3 gene.Transformants can be selected on SD medium. The the cyp52M1 gene of thePT36 strain was knocked out as described in Example 1.

Example 4.1.1 Green Fluorescent Protein

Introduction

This strain contains the yEGFP gene which was codon optimised forCandida albicans (Cormack et al., 1997). The strong constitutive GAPDpromoter from Candida bombicola (Van Bogaert et al., 2008a) was used todrive expression of the gene.

Materials and Methods

Strains, Plasmids and Culture Conditions

Escherichia coli XL10GOLD ultracompetent cells were used for plasmidmaintenance and for all cloning experiments. C. bombicola was culturedon yeast peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, 2%glucose) or on synthetic dextrose (SD) medium (0.67% yeast nitrogen basewithout amino acids (DIFCO) and 2% glucose). Liquid yeast shake-flaskcultures were incubated at 30° C. and 200 rpm. E. coli was grown inLuria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract and 0.5%sodium chloride) supplemented with 100 mg/L ampicillin if necessary.Liquid E. coli cultures were incubated at 37° C. and 200 rpm. PlasmidpGALyEGFPTU which harbours the yEGFP GFP variant, was obtained from theLaboratory of Molecular Biology, Ghent University (LMBP). This GFPvariant contains two mutations (S65G and S72A) relative to the wild typeGFP and is additionally codon optimized for the yeast Candida albicans(Cormack et al., 1997).

DNA Isolation and Sequencing

The pGEM®-T vector (Promega) was used for all cloning experiments. T4DNA ligase (Fermentas) and the In Fusion Dry Down PCR cloning Kit(Clontech) were used for cloning and ligation. All restriction nucleaseswere obtained from New England Biolabs (NEB) and restriction digestswere performed as specified by the supplier. Yeast genomic DNA wasisolated of overnight yeast cultures grown on YPD. The yeast cell wallwas enzymatically removed by incubation of the cell pellet derived from1 ml of yeast culture with 0.80 g Yeast Lytic Enzyme (Sigma)/g wet cellweight in SCE buffer (1M sorbitol, 0.1M sodium acetate and 60 mM EDTA,pH 7.5) for 90 min at 37° C. in presence of 3.75 μl mercaptoethanol.Genomic DNA was isolated from the remaining protoplasts by means of theDNeasy® Plant Maxi Kit (Qiagen). Plasmid DNA was isolated by using theQIAprep Spin Miniprep Kit (Qiagen). PCR reaction mixtures were purifiedusing the QiAquick PCR Purification Kit (Qiagen) or the column free SureClean Plus Kit (Bioline) for PCR fragments larger than 4 kb. The latterwas also used for purificationp of restriction mixtures. All DNAsequences were determined at LGC Genomics, Germany.

Primer Design and PCR Reactions

Primer design, sequence analysis and strategy design were performed withthe Clone Manager Professional Suite software (Version 8.0). Primerswere ordered at Sigma. All high fidelity PCR reactions were performedusing the Pfu high fidelity polymerase unless stated otherwise. ColonyPCR's both on E. coli and C. bombicola were performed using Taqpolymerase.

Creation of the GFP Expression Cassette

A ‘central’ vector (pGEM-T_cassette_yEGFP) was created which containedthe yEGFP gene flanked at both sides by an approximately 1 kb longsequence for recombination at the genomic P/T ura3 locus in PT mutantsof C. bombicola ATCC 22214 (described above) and additionally containsthe ura3 marker for selection. The vector was constructed in such waythat it can be cut open with two unique restriction enzymes of which one(SapI), cuts just before the ATG ‘start’ codon of the gfp gene. Thisfeature of the vector allows to clone promoter fragments exactly infront of the GFP variant. Hence, a multiple cloning site (MCS3) wasconstructed of which the sequence was added as a non binding extensionto primer P5_FOR_yEGFP_extMCS3 (bold characters in Table 6). This MCScontains 11 unique restriction sites of which two (SapI and AvaI) wereused to linearize the vector. The linearized vector was then used forcloning the GAPD promotor in front of the GFP variant using the InFusion Dry Down PCR cloning kit.

The vector pGEM-T_cassette_yEGFP was constructed in eight steps. Firstthe 5′UTR of the ura3 gene (including the ura3 promotor), the ura3coding sequence and ura3 3′UTR (including the ura3 terminator) wereamplified as one PCR fragment (1970 bp) from genomic DNA of wild type C.bombicola ATCC 22214, using primers P1_FOR_URA3v and P2_REV_URA3v. Sincethe pGEM-T vector was used for cloning the High Fidelity PCR Master Kit(Roche) was used for amplification. This first PCR fragment (PRODUCT 1)provides the final cassette with 1000 base pairs needed for homologousrecombination in PT mutants of C. bombicola, while at the same timeproviding the cassette with a selection marker. This resulting vectorpGEM-T_ura3* still contained a SapI recognition site, which wasoriginally present in pGEM-T. This recognition site needed to be removedto make SapI a unique restriction enzyme. The latter was done using theQuick Change Site Directed Mutagenesis kit (Stratagene) with themutagenesis primerpair P7_FOR_QCSapIpGEM-T and P8_REV_QCSapIpGEM-T. TheSapI recognition site ‘5-GCTCTTC-3’ was thus transformed into thefollowing sequence: ‘5-GCTCCTC-3’ which will not anymore be recognisedby the SapI restriction endonuclease rendering the SapI recognition siteof MCS3 unique. The resulting vector was named pGEM-T_ura3.

A second step involved the amplification of the 3′ UTR of the ura3 genefrom genomic DNA using primers P3_FOR_URA3t_extyEGFP andP4_REV_URA3t_extNotI (PRODUCT 2) and amplification of the yEGFP variantand Candida albicans MAL2 terminator from pGALyEGFPTU using primersP5_FOR_yEGFP_extMCS3 and P6_REV_yEGFP_extURA3t. The multi cloning siteMCS3 and a NotI restriction site were added to PRODUCT3 and PRODUCT2respectively by non binding extensions on primers P4_REV_URA3t_extNotIand P5_FOR_yEGFP_extMCS3. PRODUCT3 and PRODUCT2 were subsequently fusedtogether by overlap PCR. The template PCR products already contained thefifteen necessary complementary base pairs for performing overlap PCRwhich were added as non binding extensions on primersP3_FOR_URA3t_extyEGFP and P6_REV_yEGFP_extURA3t. Template PCR productswere cleaned up using the Qiaquick PCR Purification Kit, concentrationswere measured and three PCR reactions were set up with differenttemplate concentrations; 0.5 ng, 5 ng and 50 ng of PRODUCT2 and PRODUCT3were added to three separate PCR tubes. Subsequently, fifteen primerlessPCR cycles were conducted following the next temperature program; aninitial denaturation at 95° C. for 2 min; a 15-fold repeat of thefollowing three steps: 95° C. for 30 sec, a specific annealing at 60° C.for 30 sec and an elongation step at 72° C. for 3 min. These fifteencycles were followed by a final elongation step at 72° C. for 7 min.After this first primerless PCR, 3 μl of each of the two primers foramplification of the fusion product, P5_FOR_yEGFP_extMCS3 andP4_REV_URA3t_extNotI, was added to each of the PCR tubes. Next, 30regular PCR cycles were performed. The fusion PCR product, P3_P2 (=2275bp), was subsequently cut with SpeI and NotI as was the vectorpGEM-T_ura3. Vector and insert were subsequently ligated using T4 DNAligase and the ligation mixture was transformed into XL10Gold competentE. coli cells. The resulting vector was named pGEM-T_cassette_yEGFP.

The constructed vector was subsequently used to clone the GAPD promoterin front of the yEGFP start codon. The vector was linearized using theSapI and AvaI restriction nucleases and the In Fusion Dry Down PCRCloning Kit was used to clone the promoter into the linearized vector.For this purpose the promoter was provided with the fifteen base pairsnecessary for cloning linear PCR fragments into a linearized vector.These were added as non binding fragments on the primersP18_REV_GAPDprom and P19_FOR_pGAPD1560 used for promoter amplificationfrom genomical DNA of C. bombicola and are depicted bold in Table 6. Theconstruced vector was named pGEM-T_pGAPD1555_yEGFP.

Transformation

Yeast cells were transformed by standard electroporation. Linear DNA fortransformation was obtained by standard PCR with the primersP1_FOR_URA3v and P31_REV_cassette (Table 6). Transformants were selectedon SD plates. E. coli cells were transformed as described by Sambrook(Sambrook et al., 2001) and selection occurred on LB plates supplementedwith ampicillin.

Fluorometry

Fluorescence was measured using a Spectramax Gemini XS device (MolecularDevices, St. Gregoire, France) with black 96-well plates. Fluorescenceemission was measured at 511 nm after excitation at 488 nm and wasquantified in relative fluorescence units (RFU's). Cultures were grownon SD and before measuring fluorescence. The wild type C. bombicola ATCC22214 was uses as a blank reference.

Results

C. bombicola was transformed with the GFP expression cassette asdescribed in the material and method section. After five days ofincubation colonies appeared on the selective SD plates. Colony PCR wasperformed with the primerpair P37_FOR_checkGFP and P35_REVcheckcasIN andone positive colony was obtained. The genomical DNA of mutant E1(Candida bombicola_pGAPD1555_yEGFP) was isolated and several controlPCR's were performed to check for the correct genotype. These controlPCR's confirmed that the mutants contained the complete integrationcassettes.

Three biological replica's of the mutant and WT respectively were grownon 3 mL SD medium in deep 24-well plates at 30° C., 200 rpm. The wellswere inoculated to the same OD (0.2) from precultures on SD started fromsingle colonies. Several samples were taken throughout the growth curve.100 microliter (×2) of each well was put into a black 96 well plate andfluorescence was measured after excitation at 488 nm. Blanc valuesobtained for the wild type were substracted from the fluorescence valuesof the mutant and WT respectively (background) and the obtained valueswere plotted against the incubation time.

As can be seen in FIG. 12 a significant fluorescent signal was detectedbetween 40 and 63 hours of incubation.

Example 4.1.2 Amylase

Introduction

In order to explore its capabilities to produce (heterologous) enzymes,the sophorolipid negative Candida bombicola ATCC 22214 cyp52M1 wastested as to its production of an—amylase.—Amylases constitute animportant class of enzymes which find many biotechnological applicationsin processes which involve, for example, the degradation of starch andthe determination of soluble and insoluble dietary fiber in rice andwheat bran. Such applications are found in baking, brewing, detergentsand textile industries (Roy et al., 2000). In baking, for example,-amylases are used because they increase the bread volume, because theyimprove the crumb grain, crust, and crumb color, and for their flavordevelopment promoted in the final product (Rosell et al., 2001).The—amylase is an endo-enzyme that randomly hydrolyses the—1,4glucosidic linkages in polysaccharides.

For heterologous expression in Candida bombicola, the α-amylase fromAspergillus oryzae (amy3) has been selected. The strong constitutiveGAPD promoter from Candida bombicola (GenBank accession number EU315245)was used to drive expression of the gene. In order to get the enzymesecreted, an N-terminal secretion signal has been provided (the S.cerevisiae α mating factor secretion signal). Since codon usage of A.oryzae significantly differs from that in C. bombicola, and C. bombicolahas no multiple exon-intron genes, a codon optimized cDNA sequence hasbeen designed.

Materials and Methods

Strains, Plasmids and Culture Conditions

C. bombicola knocked-out in the cyp52M1 gene and in the ura3 gene (PT36strain) was used for amylase expression. More specific, the amylaseexpression cassette was used to transform a sophorolipid negative,auxotrophic ura3⁻ C. bombicola ‘PT mutant’ (described in theintroduction of example 4).

Escherichia coli DH5α was used in cloning experiments and for plasmidmaintenance.

Plasmid pGEM-T_pGAPD1555_yEGFP (as described in Example 4.1.1:production of the protein GFP in Candida bombicola) was used as vectorbackbone. This vector contains a bacterial selection marker (AmpR) andorigin of replication, a functional copy of the C. bombicola ura3 gene,and the yEGFP gene. Transcription of the yEGFP is controlled by 1560 bpof the C. bombicola GAPD promoter region (GenBank accession numberEU315245) and the Candida albicans MAL2 terminator (GenBank accessionnumber M94674). The promoter-yEGFP-terminator construct is flanked atboth sides by an approximately 1 kb long sequence for recombination atthe genomic PT ura3 locus in PT mutants of C. bombicola ATCC 22214,being the functional copy of the C. bombicola ura3 gene at the 5′upstream side.

For routine experiments, yeast cells were grown on YPD medium (1% yeastextract, 2% peptone and 2% dextrose), while selection aftertransformation was performed on synthetic dextrose (SD) medium (0.67%yeast nitrogen base without amino acids (DIFCO) and 2% glucose) andamylase production was performed on 3C medium with 3% sucrose (10%glucose, 1% yeast extract, 0.1% ureum, 3% sucrose). Yeast cultures wereincubated at 30° C. and 200 rpm.

E. coli cells were grown in Luria-Bertani (LB) medium (1% trypton, 0.5%yeast extract, 0.5% sodium chloride (and 15% agar for plates))supplemented with 100 mg/L ampicillin. E. coli cultures were incubatedat 37° C. and 200 rpm.

Standard DNA Manipulation

Routine recombinant DNA methodology was performed according to Sambrook& Russell (2001) (Sambrook and Russell, 2001). DNA concentrations weremeasured with the NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDropTechnologies). The 2-Log ladder of Westburg BV was used to control thelength of DNA products. PCR reactions were performed with standardTaq-DNA polymerase (Westburg BV) or with the PfuUltra™ High-Fidelity DNAPolymerase AD (Stratagene) according to the procedure described by themanufacturer. Gel fragments were purified with the Qiaexll GelPurification kit (Qiagen). Ligation was performed with the T₄ DNA ligaseof Fermentas, according to the manufacturer's protocol. Plasmid DNA wasisolated with the QIAprep Spin Miniprep Kit (Qiagen). Sequencing wasperformed at AGOWA (LGC genomics). Primer design, sequence analysis andstrategy design was done with the Clone Manager Professional SuiteSoftware (Version 8.0). All primers were obtained from Sigma-Aldrich Co.Restriction enzymes were obtained from New England Biolabs (Westburg BV)and were used as indicated by the manufacturer.

Creation of the Amylase Expression Cassette

Since codon usage of A. oryzae significantly differs from that in C.bombicola, and C. bombicola has no multiple exon-intron genes, a codonoptimized cDNA sequence was designed (FIG. 13). The protein sequence ofthe mature α-amylase from Aspergillus oryzae (TAKA-amylase A, EC3.2.1.1., encoded by amy3, Genbank accession number CAA31220.1) was backtranslated using the averaged codon usage of 10 C. bombicola genes knownto be well expressed. In the synthetic construct, the amylase codingsequence (sAmyAO) is preceded by the sequence coding for 85 amino acidsof the S. cerevisiae α mating factor secretion signal (GenBank accessionnumber NP_(—)015137), which was also back translated for codonoptimization (sMFaScss). Part of the C. bombicola GAPD promoter (GenBankaccession number EU315245) was placed 5′ upstream to the syntheticconstruct (pGAPD). Finally, some mutations were made to introduce amulti cloning site in front of the ATG start codon and at the 3′ end ofthe coding sequence. The final sequence of the ordered construct isgiven in FIG. 14. The construct (2153 bp) was ordered as such atGenScript (Piscataway, USA) and obtained cloned at the EcoRI site inplasmid cloning vector pUC57.

The synthetic amylase expression cassette was isolated from the pUC57 byrestriction with BssHII and BsiWI. The vector backbonepGEM-T_pGAPD1555_yEGFP was cut with BssHII and BsrGI, creatingcompatible ends. After gel extraction, the amylase expression cassettewas ligated in the pGEM-T_pGAPD1555_yEGFP vector backbone, resulting invector p_sAmyAO_pGapd_iUra (FIG. 15). The ligation mixture wastransformed in E. coli Dh5, and positive colonies were selected bycolony PCR with primers sAmyAOfw and sAmyAOry (Table 7). The sequence ofthe constructed plasmid was confirmed by sequencing at Agowa (LCGGenomics). Plasmid p_sAmyAO_pGapd_iUra is the same aspGEM-T_pGAPD1555_yEGFP, but the yEGFP coding region has been replaced bythe amylase coding region (and secretion signal).

Prior to transformation, the amylase expression cassette together withthe ura3 marker (6426 bp) was linearised from the p_sAmyAO_pGapd_iUraplasmid by restriction with BglI and EagI. The linearised cassettecontained regions for homologous recombination at the ura3 locus,approximately 1 kb long (FIG. 15).

Yeast Transformation and Verification of Transformants C. bombicolacells were transformed with the lithium acetate method (Gietz et al.,1995), but 50 mM LiAc was used instead of 100. Transformants wereselected on synthetic dextrose (SD) plates (uracil prototrophy restoredwith C. bombicola ura3).

Yeast colony PCR was performed with Taq DNA polymerase (Westburg BV),according to the manufacturer's protocol, with the exception thatinitial denaturation was performed for 7 minutes.

Amylase Enzyme Test

Samples of 200 μL were taken from the 3C+3% sucrose cultures. After ODmeasurement, the samples were pelleted by centrifugation at 12000 rpmduring 3 minutes. The supernatant was used for enzyme essays with theamylase test kit AMYL® (Roche Diagnostics).

The enzyme test mixture contained 1 μL sample, 50 μL R₁, 10 μL R₂, and139 μL citrate-phosphate buffer, pH 7.0. The mixture is incubated in thespectrophotometer at 37° C. The absorbance is measured every 30 s during30 minutes.

One unit amylase is defined as the amount of enzyme able to raise theOD_(405 nm) by 1 during 15 minutes in a reaction mixture consisting of 1μL sample+50 μL R₁+10 μL R₂+139 μL buffer incubated at 37° C.

Results

The amylase expression cassette, together with the ura3 marker, wasconstructed as described in the “Materials and Methods” section. Thislinear fragment (6426 bp) was used to transform sophorolipid anduracil/uridine negative Candida bombicola PT cells. One colony appearedon the selective SD plates after 4-10 days of incubation. The genotypeof this transformant was checked by yeast colony PCR with 2 primerpairs. First, primers sAmyAOfw and sAmyAOry (Table 7) were used, bothannealing in the amylase encoding region of the cassette. In this way,integrity of the amylase expressing part was ensured. The second primerpair used was sAmyAOfw2 and P35 (Table 7). Primer sAmyAOfw2 binds in theamylase encoding region, while the latter binds outside the homologousregions of the cassette, on genomic DNA. Thus, correct integration ofthe cassette at the ura3 locus is checked. The colony showed the correctgenotype and is further referred to as ‘SL⁻Amy⁺’.

The ‘SL⁻Amy⁺’ transformant and the wild type were subsequently grown on3C medium with 3% sucrose. The amylase production, cell growth (OD 600nm) and pH of the cultures were followed during 48 h, taking samplesapproximately every 2 hours. Amylase enzyme tests were performed asexplained in the “Materials and Methods” section. The results of thisgrowth and production test are depicted in FIGS. 16 and 17. From FIG.16, it is clear that the ‘SL⁻Amy⁺’ transformant produces amylase, up to106 units/μL culture supernatant, while the wild type strain does notproduce any amylase (9 units/μL maximum, background). The growth and pHcurves (FIG. 17) are similar for both strains, though the pH drops lowerin the ‘SL⁻Amy⁺’ transformant culture at the very end of the test.Amylase production has no negative effect on the growth of C. bombicola.In conclusion, an amylase expression transformant was created which isknocked out in sophorolipid production but instead produces -amylase, upto 106 units/μL culture supernatant when grown on 3C medium with 3%sucrose. The wild type strain did not produce any amylase. This is thefirst time (extracellular) enzyme production is described for asophorolipid negative strain of C. bombicola.

Example 4.2 Creation of a Strain Synthesising Polyhydroxyalkanoates

Introduction

This strain is knocked-out in the cyp52M1 gene (GenBank accession numberEU552419) mentioned above and instead carries a PHA synthase (phaC1)gene of which expression depends on the up- and downstream regulatorysequences of the cyp52M1 gene. The protein sequence of PHAC1_(pr) (ENAaccession number AAD26365.2) from Pseudomonas resinovorans wasbacktranslated using the averaged codon usage of the genes of thesophorolipid pathway. The resultant strain does not producesophorolipids anymore but instead produces MCL-polyhydroxyalkanoates(PHA) when grown on glucose with addition of rapeseed oil. Cell growthand viability of said strain is comparable to the wild type.

Materials and Methods

Strains, Plasmids and Culture Conditions

Candida bombicola ATCC 22214 was used for isolation of the ura3 genewith up- and downstream regulatory sequences and for isolation of thedownstream region of the cyp52M1 gene (downCYP). The auxotrophic ura3⁻Candida bombicola PT36 strain (which is described under Example 4.1.1)was used for insertion of the PHA expression cassette. Yeast cells weregrown on YPD medium (1% yeast extract, 2% peptone and 2% dextrose), SDmedium (0.67% yeast nitrogen base without amino acids (Difco), 2%glucose), 3C medium (10% glucose, 1% yeast extract, 0.1% ureum and 15%agar) or the medium described by Lang (Lang et al., 2000) to whichrapeseed oil (Sigma) was added after 48 h when mentioned. Yeast cultureswere incubated at 30° C. and 200 rpm.

Escherichia coli DH5α or XLI OGOLD were used in the cloning experimentsand transformation occurred as described by Sambrook and Russell (2001).E. coli cells were grown in Luria-Bertani (LB) medium (1% trypton, 0.5%yeast extract, 0.5% sodium chloride (and 15% agar for plates))supplemented with 100 mg/L ampicillin. Liquid E. coli cultures wereincubated at 37° C. and 200 rpm.

DNA Isolation and Sequencing

Yeast genomic DNA was isolated with the GenElute™ Bacterial Genomic DNAKit (Sigma) of overnight yeast cultures grown on YPD. The yeast cellwall was enzymatically removed by incubation of the cell pellet derivedfrom 1 ml of yeast culture with 0.80 g Yeast Lytic Enzyme (Sigma)/g wetcell weight in SCE buffer (1M sorbitol, 0.1M sodium acetate and 60 mMEDTA, pH 7.5) for 90 min at 37° C. in presence of 3.75 μlmercaptoethanol. Genomic DNA was isolated from the remaining protoplastsby means of the GenElute™ Bacterial Genomic DNA kit (Sigma).

Bacterial plasmid DNA was isolated with the QIAprep Spin Miniprep Kit(Qiagen). All PCR products were cloned into the pGEM-T® vector (Promega)or derivatives of it and send as such to AGOWA (LGC genomics) forsequence analysis. The pGEM-T® vector was the backbone of allconstructed vectors. When necessary DNA was isolated from gel using theQIAquick Gel Extraction Kit (Qiagen).

Primer Design and PCR Reactions

Primer design, sequence analysis and strategy design were performed withthe Clone Manager Professional Suite software (Version 8.0). Primerswere ordered at Sigma. All high fidelity PCR reactions were performedusing the Pfu high fidelity polymerase. Colony PCR's both on E. coli andC. bombicola were performed using Taq polymerase.

Transformation

C. bombicola cells were transformed using a standard electroporationprotocol. Transformants were selected on SD plates. E. coli cells weretransformed as described by Sambrook (Sambrook et al., 2001) andselection occurred on LB plates supplemented with ampicillin.

Synthetic Construct

The PHAC1 protein sequence from Pseudomonas resinovorans (ENA accessionnumber AAD26365.2) was backtranslated using the average codon usage ofthe genes of the SL-pathway which was determined using an online tool(Stothard, 2000). An SKL (TCTAAGCTG) peroxisomal target sequence (PTS)was added at the 3′ terminus of the gene as well as the up- anddownstream regulatory regions of the cyp52M1 gene respectively at the 5′(488 bp) and 3′ (190 bp) side of the codonoptimised phac1 sequence. The5′ UTR regio was extended to 1098 bp to obtain enough homology forhomologous recombination at the cyp52M1 locus. The construct was orderedas such at GenScript (Piscataway, USA) and obtained cloned in a vector.The construct was amplified with the primers P55_FOR_upCYP_extNheI andP58_REV_PHAC1+tCYP_extEcorI (Table 8) yielding a fragment of 2986 bp.The primers respectively contained NheI and EcorI extensions so that thefragment could be subsequently digested with said restriction enzymesfor further subcloning of the synthetic construct.

Creation of the PHA Expression Cassette

The expression cassette contained the codon optimised phaC1 gene andura3 marker and was constructed in such way that it contained the up-and downstream regio's of the cyp52M1 gene for homologous recombinationat the cyp52M1 locus in the genomic DNA of an auxotrophic C. bombicolaPT36 strain. Construction of the cassette occurred in three steps. Firstthe region for homologous recombination at the 3′ end of the cyp52M1gene (downcyp) was amplified from genomic DNA of C. bombicola usingprimers P53_FOR_downCYP_extSpeI and P54_REV_downCYP_extNotI (Table 8).The resulting amplicon and the pGEM-T®_ura3 vector (cfr. Example 4.1.1)were digested with the unique cutters SpeI and NotI and subsequentlyligated using T4 ligase (NEB). Secondly the resulting vector wasdigested using the unique cutters NheI and EcorI. This doublerestriction yielded two fragments (5644 bp and 358 bp) of which thelargest one was gel purified and subsequently ligated with the amplifiedsynthetic construct which was first subjected to restriction with thesame restriction enzymes. In a third and last step the expressioncassette was amplified using primers P63_FOR_cassPHAC1 andP64_REV_cassPHAC1 and the linear PCR fragment was purified and used fortransformation of the C. bombicola PT36 strain (FIG. 18). Transformantswere selected on SD plates.

Sampling

Analytical sophorolipid samples were prepared as follows: 440 μLethylacetate and 11 μL acetic acid were added to 1 mL culture broth andshaken vigorously for 5 min. After centrifugation at 12000 rpm for 5min, the upper solvent layer was removed and put into a fresh eppendorftube with 700 μL ethanol. Samples were analysed by HPLC and EvaporativeLight Scattering Detection.

Cell dry weight (CDW) was measured by transferring 2 mL culture broth toa cellulose nitrate filter with a pore diameter of 0.45 μm (Sartorius)and the dry weight was determined in the XM60 automatic oven fromPrecisa Instruments Ltd.

PHA hydrolysis and fatty acid methyl ester (FAME) formation wasperformed by performing acid methanolysis of 30 mg of freeze-dried cellmaterial in a 4 ml 1:1 chloroform/methanol+3% H₂SO₄ mixture at 95° C.for 4 h. Before methanolysis cells were harvested (4000 rpm, 4° C.) fromflask cultures after which the cell pellets were frozen at −80° C. andlyophilised for 24 h. 30 mg of the resulting freeze-dried material waswashed several times with 25 ml of hot methanol (65° C.) to remove oiland free fatty acids. After methanolysis 4 ml 0.9% (wt/vol) NaCl wasadded to the tubes and the organic phase was collected for analysis onGC-MS. 1 mg of internal standard (2-hydroxyhexanoic acid) was addedbefore methanolysis and 1 mg of external standard (12-hydroxydodecanoicacid) was added just before injection of the samples.

HPLC-Analysis of Sophorolipids

Sophorolipid samples were analysed by HPLC on a Varian Prostar HPLCsystem using a Chromolith® Performance RP-18e 100-4.6 mm column fromMerck KGaA at 30° C. and Evaporative Light Scattering Detection(Alltech). A gradient of two eluents, a 0.5% acetic acid aqueoussolution and acetonitril, had to be used to separate the components. Thegradient started at 5% acetonitril and linearly increased to 95% in 40min. The mixture was kept this way for 10 min and was then brought backto 5% acetonitrile in 5 min. A flow rate of 1 mL/min was applied.

GC-MS Analysis of FAMES

The GC (TraceGC ultra, Interscience) contains a 0.25 mm Rxi®-1 ml column(Restek) which is coated with dimethyl polysiloxane. The used carriergas was helium. The following temperature profile was used: 2 minutes at64° C. followed by a linear increase of 30° C./min to 200° C. When thecolumn reached 200° C. a second linear increase of 50° C./min to 310° C.took place. De eluting compounds were subsequently injected into the MS(DSQ, Interscience) where they were ionized and detected for furtheridentification. The latter was done using the Xcalibur software whichwas coupled to the NIST MS Search 2.0 bibliotheca. Only compoundsbetween 40 en 650 g/mol were detected.

Results

The PHA expression cassette was constructed as described in the“Materials and Methods” section. This linear fragment was used totransform Candida bombicola PT36 cells (FIG. 18). 16 colonies appearedon the selective SD plates after 4-11 days incubation. The genotype ofthese 16 transformants was checked by yeast colony PCR with the primerpair P9_FOR_seqQCSapI_URA3down binding on the expression cassette andA21totRev, binding on the genomic DNA downstream of the rightrecombination site (Table 8). Ten of the colonies showed the correctgenotype. Two other colony PCR's were performed on the positive coloniesto control for correct insertion of the cassette. Genomical DNA of onemutant (A8) was subsequently isolated and a PCR reaction was performedwith primer pair UDPGTA1R and A21TotRev binding on the genomic DNA of C.bombicola just up- and downstream of the left and right recombinationsites respectively. This PCR fragment was sent for sequencing andanalysis revealed that the PHA expression cassette was correctly andcompletely integrated at the cyp52M1 locus of C. bombicola and containedno errors.

The A8 mutant was subsequently grown on the medium described by Lang.Three biological replicas were supplemented with rapeseed oil after 48 hof incubation, three others weren't. Each shake flask was inoculatedfrom a different preculture from overnight grown cultures on Lang medium(5 mL) inoculated from one colony of a 3C plate. The wild type was grownin parallel as a control. Samples for SL extraction were takenthroughout the growth curve as were samples for glucose consumption andCDW determination. Thirteen days after addition of the oil the cellswere harvested and PHA hydrolysis and conversion to FAMES was performedas described in the “Material and Methods” section.

The effect of the disruption of the cyp52M1 gene was similar as thisdescribed in Example 1. CDW and growth were similar for the PHAC1expression mutant and the wild type and glucose consumption instationary phase was slower for the PHAC1 expression mutant. Whereasthere clearly was sophorolipid production for the wild type, nosophorolipids could be detected in the medium for the PHA expressionmutant with or without addition of oil.

GC-MS analysis of the FAMES derived from end samples of the growthexperiment of the PHAC1 A8 mutant (see materials and methods) to whichrapeseed oil was added led to identification of compounds derived fromMCL-PHA produced in the Candida bombicola PHAC1 A8 mutant (FIG. 19).Three compounds derived from PHA were detected in two of the threebiological replicas: 3-methylhydroxyoctanoate (0.50% wt/dwt),3-methylhydroxydecanoate (0.54% wt/dwt) and 3-methylhydroxydodecanoate(0.32% wt/dwt) with a total of 1.36 wt/dwt PHA. In the third biologicalreplica 3-methylhydroxytetradecanoate was additionally detected (0.50%wt/dwt) and the amounts of the other PHA-monomers were slightly higherfor this flask: 3-methylhydroxyoctanoate (0.64% wt/dwt),3-methylhydroxydecanoate (0.73% wt/dwt) and 3-methylhydroxydodecanoate(0.29% wt/dwt) with a total amount of 2.16% wt/dwt PHA. These peaks werenot detected for the samples derived from the wild type cultures (withand without addition of rapeseed oil) nor for the samples from the PHAC1A8 mutant to which no rapeseed oil was added. Hence the peaks were notderived from intermediates of the beta-oxidation which were converted toFAMES during methanolysis. The rapeseed oil was needed as a lipogenicsource to produce the PHA (in substantial amount). The amount ofproduced PHA was quantified using an internal standard(2-hydroxyhexanoic acid)) which was added to the samplesbeforemethanolysis.

The cyp52M1 regulatory sequences drove expression of the PHAC1 gene inthis experiment. It was further found that a substantial glucoseconcentration, in combination with N or P starvation, is needed toactivate this promoter. Glucose on the other hand represses expressionof the genes of the beta-oxidation. To obtain higher PHA production thecatalase (pCTA) and isocitrate lyase (pICL) promoters are isolated fromthe genome and PHAC1 expression is derived from these promoters whichare repressed by high glucose concentrations. Disruption of thebeta-oxidation and feeding with substrates with long (C16 and C18) chainlength leads to the production of PHA composed only of C18 and/or C16monomers.

In conclusion, a PHAC1 expression mutant was created which is knockedout in sophorolipid production but instead produces MCL-PHA up to 0.99wt/dwt when grown on glucose with addition of rapeseed oil.

Example 4.3 Production of Glycolipids Example 4.3.1 Glucolipids

Due to the free carboxyl group, an unsaturated carbon chain and thepresence of a carbohydrate head group, glucolipids are interestingintermediates for several kinds of biocatalytic or chemical conversionreactions. Since small structural variations can have a significantinfluence on biological activity or physico-chemical properties of aglycolipid, enzymatic or chemo-enzymatic synthesis of sophorolipidderivatives has been subject of some research papers (Bisht et al.,1999; Carr and Bisht, 2003; Rau et al., 2001) and patent applications(WO2004/044216 and US05/0164955). To date, glucolipids with a freecarboxylic end are only produced by enzymatic conversion of acidic(open-ring) sophorolipids which are on their turn obtained afteralkaline hydrolysis of the crude C. bombicola bioproduct (Rau et al.,1999; Saerens et al., 2009). On the other hand, alkyl glucosides couldbe obtained by microbial conversion of secondary alcohols (Brakemeier etal., 1998) or branched fatty alcohols (Palme et al., 2010). Acidicglucolipids (and sophorolipids) especially attract attention becausethey are asymmetrical bolaamphiphiles that, in addition to thesupramolecular structures they typically form, also have increasedchemical versatility as compared to the chemically synthesizedsymmetrical ones (Zhou et al., 2004). The ΔugtB1 deletion mutant createdin Example 3 is an interesting strain that now offers time-saving invivo production of these biomolecules starting from cheap renewablesubstrates. When we repeated the flask fermentation with the B11 mutantbut prolonged the incubation on rapeseed oil to 14 days, the producedglucolipids were identified to be a mixture of structurally relatedmolecules as revealed by mass spectrometric analysis of culture extracts(see material and method section Example 3 and FIG. 20). In addition tothe most predominant mono-acetylated acidic glucolipids (m/z=502), minoramounts of unacetylated (sub-) terminally hydroxylated glucolipids couldbe identified (m/z=460) (FIG. 20). So far, no lactonization ofglucolipids was observed. The appearance of acetylated glucolipidsillustrates that the acetyltransferase, which normally decorates de novosophorolipids at their 6′ and/or 6″ positions with acetylgroups, showsactivity towards glucolipids as well. In this respect it is possiblethat acetylation of glucolipids occurs before addition of the secondglucosyl unit during de novo sophorolipid synthesis without beingnecessary for this second glucosylation reaction.

After 14 days incubation on rapeseed oil, residual oil still floats ontop of the B11 culture while a wildtype fermentation after this timecompletely utilized rapeseed oil for sophorolipid production with acommon yield around 50 g/L, indicating less efficient substrateconversion to glucolipids as compared to sophorolipids.

The production of glucolipids by the ΔugtB1 deletion mutant now createsa time efficient in vivo production process of these interestingglycolipid intermediates and this by conventional fermentation on cheapsubstrates.

Example 4.3.2 Cellobioselipids

Introduction

Cellobioselipids are produced in nature by several yeasts such asCryptococcus (Puchkov et al., 2002), Pseudozyma (Kulakovskaya et al.2005) and Sympodiomycopsis (Kulakovskaya et al., 2004), and thedimorphic fungus Ustilago maydis (Spoeckner et al., 1999, Teichmann etal., 2007). Their overall structure is comparable to sophorolipids,however the two glucose units are linked by a β-1,4 linkage, the fattyacid tail can show multiple hydroxylations (α-, ω- and ω-1) and thecellobiose molecule is acetylated and/or acylated with short chain(C6/C8) β-hydroxy fatty acids. Though cellobioselipids are promisingantimicrobial agents, there is to date no industrial production due tothe overall very low yields. We here create a C. bombicola mutantproducing cellobioselipids instead of sophorolipids by changing thewildtype UGTB1 gene (see example 3) by either U. maydis UGT1 orClostridium stercorarium CepB gene (Reichenbecher et al., 1997). TheUgt1 glucosyltransferase from U. maydis acts in a very comparable way tothe UgtB1 glucosyltransferase from C. bombicola, using the sameUDP-glucose as a glucosyl donor and a comparable glucolipid as anacceptor, linking both by a β-1,4 linkage instead of the β-1,2 formed bythe UgtB1 glucosyltransferase (Teichmann et al., 2007). Thebidirectional cellodextrin phosphorylase (CepB) from the cellulolyticthermophile bacterium Clostridium stercorarium is involved in cellulosedegradation but the synthetic direction of the enzyme can use(17-O-β-D-glucopyranosyl)-octadecenoic acid obtained from C. bombicolasophorolipids (Saerens et al., 2009) as an acceptor for cellobioselipidformation, using glucose-1-Pi as a glucosyl donor.

Materials and Methods

Strains and Plasmids

C. bombicola G9 (derived from ATCC 22214, see Van Bogaert et al., 2008)was used for creation of the cellobioselipid producing mutant. Yeastcells were grown on YPD medium containing 1% yeast extract, 2% peptoneand 2% dextrose, SD medium containing 0.67% yeast nitrogen base withoutamino acids (Difco) and 2% glucose or 3C medium containing 10% glucose,1% yeast extract and 0.1% urea. Liquid media were incubated at 30° C.and 200 rpm. E. coli was grown on Luria Bertani medium (0.5% Bacto yeastextract, 1% Bacto Trypton, 0.5% NaCl) containing 0.01% ampicillin andincubated at 37° C. and 200 rpm. Plasmids were isolated from E. coli bymeans of the MiniPrep Plasmid Isolation kit from Qiagen and sequenced atLGC genomics (Germany).

Creation of the UGT1 and CepB Expression Cassettes

Both UGT1 and CepB expression cassettes were created by restrictionenzyme mediated coupling of the wildtype C. bombicola UGTB1 promotor anda suitable terminator to the UGT1 and CepB genes respectively, followedby coupling of the promoter-gene-terminator sequence to the URA3selectable marker. As a terminator, both the wildtype C. bombicola UGTB1terminator as well as the tyrosine kinase (TK) terminator were used. AllPCR reactions were performed with the PfuUltra High Fidelity PCR system(Stratagene) unless stated otherwise. As a first step, the UGTB1promotor (P) in front of the UGT1 or CepB gene was amplified fromplasmids pG_PUgt1T and PG_PCepBT respectively, pGEM_T® derived plasmidsharboring the gene of interest in between the native UGTB1 promotor andterminator sequences. Primer pairs were MDR505Rev and Ugt1 1737Rev_SalIfor the 2736 bp PUgt1 fragment and MDR505Rev and CepB 2355Rev_SalI forthe 3354 bp PCepB fragment (Table 9). The 134 bp TK terminator wasamplified from a plasmid containing the hygromycin resistance marker inbetween tyrosine kinase (TK) terminator and C. bombicola GAPD promoter(Van Bogaert et al., 2008) making use of primers TK F_SalI and TK R_MluI(Table 9). All obtained fragments were purified by means of the QiaquickPCR purification kit (Qiagen) and subjected to an overnight SalI digest(New England Biolabs) according to Sambrook (Sambrook and Russell, 2001)but with a secondary addition of 20U restriction enzyme after 2 hours ofincubation. After purification of the digested fragments by means of theMinElute reaction clean-up kit from Qiagen, both PCepB and PUgt1fragments were ligated to the digested TK terminator. For that, 2U T4DNA ligase from fermentas was added to 100 ng of the PCepB and PUgt1fragments and a suitable amount of purified TK fragment was added suchthat a gene:terminator ratio of 3:1 was obtained. The mixture wasincubated overnight at 22° C. Subsequently, the ligation products wereamplified from the reaction mixtures by means of primers MDR505Rev andTKR_MluI yielding the 2856 bp PUgt1TK and 3474 bp PCepBTK fragmentrespectively. After purification, the fragments were cloned into thepJET1.2/blunt Cloning Vector using the CloneJet™ PCR Cloning kit fromFermentas and resulting plasmids pJ_PUgt1TK and pJ_PCepBTK were used fortransformation of E. coli Fusion Blue and XL10Gold competent cellsrespectively, according to Sambrook (Sambrook and Russell, 2001).Correct transformants were identified by colony PCR and the derivedplasmids were sent for sequencing. To couple the URA3 selectable marker,the inserts of plasmids pJ_PUgt1TK and pJ_PCepBTK were amplified againby means of the primers MDR505Rev and TKR_MluI, purified and subjectedto an overnight MluI digest (New England Biolabs) according to Sambrook(Sambrook and Russell, 2001) but with a secondary addition of 20Urestriction enzyme after 2 hours incubation. Accordingly, the URA3selectable marker in addition to the C. bombicola UGTB1 3′ end wasamplified from plasmid pG_KOugtB1 (see Example 3) by means of primersURA3 677F_MluI (Table 9) and GTII +239Rev (Table 4), purified andsubjected to the same overnight MluI digest. After purification of thedigested PUgt1TK, PCepBTK and URA3GT2T fragments, the genes of interestwere coupled to the selection marker. For that, 100 ng of the PUgt1TKand PCepBTK fragment respectively was added to an appropriate amount ofURA3GT2T fragment such that a gene:marker ratio of 3:1 was obtained andligation was performed overnight at 22° C. with 2U of T4 DNA ligase(Fermentas). The final ligation products PUgt1TK_URA3GT2T (5856 bp) andPCepBTK_URA3GT2T (6474 bp) were amplified from the ligation mixtures bymeans of the Expand Long Template PCR System from Roche and primersMDR505Rev and GTII +239 Rev.

Alternatively, to create expression cassettes were the gene of interestis followed by the native C. bombicola UGTB1 terminator instead of theTK terminator, the complete inserts of plasmids pG_PUgt1T and PG_PCepBTwere amplified by means of primer pair MDR505Rev/PCepBT214 R_MluIyielding the 2967 bp PUgt1T and the 3595 bp PCepBT fragmentsrespectively. The URA3 selectable marker followed by the UGTB1 3′ endbut without terminator sequence, was amplified from plasmid pG_KOugtB1with primers URA3677 F_MluI and GTII+1296Rev (Table 9). PCR productswere purified, digested overnight with MluI and ligated as describedabove. Expression cassettes PUgt1T_URA3GT2 and PCepBT_URA3GT2 wereamplified from the ligation mixtures by means of the Expand LongTemplate PCR System from Roche using primers MDR505Rev and GTII+1296Rev.All four obtained expression cassettes were gel purified using theQiaquick Gelextraction kit from Qiagen and are cloned in pGEM-T® makinguse of the pGEM-T® Vector System (Promega). Plasmids are used fortransformation of E. coli ultracompetent cells and correct transformantsare identified by means of colony PCR. The derived plasmids are sent forsequencing.

Creation of the Cellobioselipid Producing Mutant

Linear expression cassettes PUgt1TK_URA3GT2T, PCepBTK_URA3GT2T,PUgt1T_URA3GT2 and PCepBT_URA3GT2 are amplified from the pGEM-T® derivedplasmids making use of the primer MDR505Rev in combination with GTII+239Rev or GTII+1296Rev, depending of the presence or absence of theUGTB1 terminator at the 3′ end of the cassette. The cassettes arepurified and used for transformation of the ura3 deficient C. bombicolaG9 strain by electroporation. For that, C. bombicola G9 is grownovernight in 100 mL YPD and when OD reaches 1, cells are harvested from50 mL by centrifugation during 5 min at 4° C. and 4300 g. After washingtwice with cold and sterile mQ water, cells are resuspended in 2 mLsterile sorbitol solution (1 M). After centrifugation at 4° C. and 4300g, cells are resuspended in 2 mL sterile lithiumacetate (0.1 M) inpresence of 2.5 mM DTT and left to rest at room temperature for 10 to 15min. Cells are then harvested again and washed with 2 mL sorbitol (1M)before resuspending in 250 μl sorbitol (1M). From this suspension, 50 μlis carried over into a sterile eppendorf tube, 500 ng-1 μg of the linearexpression cassette is added and the mixture is incubated on ice for 2min before transfer to a 2 mm electroporation cuvette. A pulse of 1.5 kV(200 Ohm) is given during 5 milliseconds and 1 mL of cold and sterileYPD is added immediately. The cells are incubated for 1 h at 30° C. and200 rpm and harvested by centrifugation at room temperature during 5 minat 1500 g. Cells are then resuspended in 1 mL sorbitol (1M) and aliquotsof 200 μl are spread on selective SD plates. Plates are incubated at 30°C. until transformant colonies appear.

Characterization of Cellobioselipid Producing Mutants

Mutant colonies are first checked for correct integration of theexpression cassette at the UGTB1 locus of the genome by means of yeastcolony PCR. Correct transformants, appearing from double cross-overevents, are transferred to 3C medium containing 10% glucose, 1% yeastextract, 0.1% urea and 2% agarose before inoculation to liquidproduction medium described by Lang et al. (2000) in order to check forglycolipid production. Liquid media are incubated at 30° C. and 200 rpmfor two days before addition of rapeseed oil (37.5 g/L). Wildtype C.bombicola ATCC 22214 serves as a reference. One week after addition ofrapeseed oil, glycolipids are extracted from 1 ml culture samples bymeans of 400 μl ethylacetate in presence of 10 μl acetic acid.Ethylacetate fractions are analysed on HPLC-ELSD and LC-MS as describedin example 3. To verify the molecular structure of the producedcellobioselipids, the extracts of the mutants are scanned for compoundswith molecular mass in the range of 600-800 (m/z).

TABLE 1 Primers used for knocking-out the C. bombicola CYP52M1 gene. All primers were obtained from Sigma Genosys. Seq ID Name FeatureSequence NO: A21TotFor cloning CTGAGTGATAGGTTGAGCATTAG 5 CYP52M1A21TotRev cloning GCTCTTGTTCGGTACTCTTATTG 6 CYP52M1 GHlnfA21Forligating selection GCTAAAGTTACCCGA- 7 marker intoCCAATGGCAGTGGCTTACCACTC CYP52M1 HygrolnfA21Rev ligating selectionGATCCTTCTGCTCGG- 8 marker into CCCGCGTTTATGAACAAACGACCC CYP52M1A21KnockHygroCasFor amplification GAGTCGGGCGTTATTTCTCC 9 knock-outfragment A21KnockHygroCasRev amplification AATCCCATAAACGACTACTC 10knock-out fragment HygroInsertCheckFor checking knock-TTCGACAGCGTCTCCGACCT 11 out genotype Ura3outEndfor checking Cm2TAAAGAAACGAAGGGCCCAGCAGTC 12 genotype ATqRev checking Cm2CACCACAGTACGAGGAGGAACA 13 genotype

TABLE 2Primers used for isolation of the UGTA1 gene and construction of  the knock-out cassette. All primers were obtained from Sigma Genosys.Seq ID Name Feature Sequence No. UDPGTA1 3′ primary GSP 5′CAGCAGAGACCATCTGCCTACAACTTC 3′ 14 DS GSP1 primer UDPGTA1 3′ nested GSP5′ CAACGCCCAAGCACCGAACTCAATTCAC 3′ 15 DS GSP2 primer UDPGTA1High fidelity 5′ GAAGATACGTCCGTGCTTTG 3′ 16 TotF forward primer A1PHigh fidelity 5′ CATGGCTAGCCGGGCATTATATGGCCTG 3′ 17 RevNheIreverse primer A1T High fidelity 5′ CATGGCTAGCCGCTATGAACCACGCTCTTG 3′ 18ForNheI forward primer A1T Rev High fidelity 5′ CATGACAGCCTTTTCTTCTT 3′19 reverse primer Ura3 High fidelity 5′CATGGCTAGCCTGACGGGCGGATAGTACAG 3′ 20 FbisNheI forward primer Ura3High fidelity 5′ CATGGCTAGCGTCATCAACTCCATGGCGTGAGG 21 RbisNheIreverse primer 3′

TABLE 3 Ten best homology scores for the translated UGTA1 sequence NCBIE- Gene Organism Acc. N° % Id score Glycosyltransferase MycobacteriumYP_95481 35 8e−55 family protein vanbaalenii 7 UDP- MycobacteriumYP_00175 35 2e−54 glucuronosyl/ radiotolerans 4776 glucosyltransferaseGlycosyltransferase Mycobacterium gilvum YP_00113 33 2e−53 familyprotein 3857 Glycosyltransferase Mycobacterium sp. YP_64073 32 2e−52 MGTfamily 6 Glycosyltransferase Mycobacterium sp. YP_00107 32 3e−52 MGTfamily 1846 UDP- Mycobacterium YP_95566 34 4e−52 glucuronosyl/vanbaalenii 5 glucosyltransferase UDP- Acidovorax avenae YP_97296 313e−50 glucuronosyl/ 8 glucosyltransferase UDP- Burkholderia ambifariaYP7_7811 31 3e−47 glucuronosyl/ 9 glucosyltransferase UDP- Methylocellasilverstris YP_00236 31 1e−46 glucuronosyl/ 4149 glucosyltransferaseUDP- Mycobacterium gilvum YP_00113 32 2 glucuronosyl/ 3117glucosyltransferase

TABLE 4Primers used for isolation of the UGTB1 gene and construction of the knock-out cassette. All primers were obtained from Sigma Genosys.Seq ID Name Feature Sequence No. GTII −472For High Fidelity forward 5′GAGAGTGGGACCTGATTC 3′ 22 primer GTII +239Rev High Fidelity reverse 5′CTGCTCTCAACACCGAGTGTAG 3′ 23 primer Ura3inf High Fidelity forward 5′ 24ugtB1 F primer AAGCAGAGAAGGCGCGATAGTACAGGCTTT GCC 3′ Ura3infHigh Fidelity reverse 5′ 25 ugtB1 R primerCCTTCGTGGCCCCGATCATCGTCACTATAC ACATCG 3′ KOugtB1 Control forward primer5′ AAGCCAAAATCAGAGAGTG 3′ 26 Ctrl F KOugtB1 Control reverse primer 5′GGTTCTGCGAAACTGGTATG 3′ 27 Ctrl R

TABLE 5 Ten best homology scores for the translated UGTB1 sequence NCBIAcc. E- Gene Organism N° % Id score UDP- Mycobacterium YP_001754 321e−15 glucuronosyl/ radiotolerans 776 glucosyltransferase UDP-Burkholderia ambifaria YP_778119 31 5e−15 glucuronosyl/glucosyltransferase Glycosyltransferase Mycobacterium YP_954817 32 2e−14family protein vanbaalenii UDP- Acidovorax avenae YP_972968 28 1e−13glucuronosyl/ glucosyltransferase UDP- Methylocella silverstrisYP_002364 28 2e−13 glucuronosyl/ 149 glucosyltransferaseGlycosyltransferase Pectobacterium YP_003258 29 1e−12 MGT familywasabiae 26 Glycosyltransferase Mycobacterium gilvum YP_001133 31 2e−11family protein 857 Glycosyltransferase Methylobacterium YP_002500 281e−10 family 28 nodulans 506 Glycosyltransferase PectobacteriumYP_003018 28 2e−10 MGT family carotovorum 782 GlycosyltransferaseMycobacterium sp. YP_001071 26 7e−10 MGT family 846

TABLE 6 Primers used for creating pGEM-T_cassette_yEGFP. Seq ID NameFeature Sequence No. P1_FOR_URA3v Pick up AGAACAAGGCCGAGTATGTC ura3 gene+5′ UTR P2_REV_URA3v Pick up TGCCAGCAGATCATCATCAC 29 ura3 gene +3′ UTRP3_FOR_URA3t_exty Overlap GGATCCCCGCAGGGCATGCAACTTGCACATGAA 30 EGFPprimer TACC ura3- yEGFP P4_REV_URA3t_ AmplificationTAGCGGCCGCGTCAGATTAGCCTCCGACATAG 31 extNOTI ura3- yEGFP P5_FOR_yEGFP_Amplification GCACTAGTATACCCGGGCGCCT- 32 extMCS3 ura3-CAGCTCTTCGATGTCTAAAGGTGAAGAAT yEGFP P6_REV_yEGFP_ OverlapTATTCATGTGCAAGTTGCATGCCCTGCGGGGAT 33 extURA3t primer CCATACG ura3- yEGFPP7_FOR_QCSaplpGEM-t Mutagenesis CGTATTGGGCGCTCCTCCGCTTCCTCGCTCACTG 34primer ACTC P8_REV_QCSaplpGEM-t MutagenesisGAGTCAGTGAGCGAGGAAGCGGAGGAGCGCCC 35 primer AATACG P18_REV_GAPDpromPick up TTCACCTTTAGACATTTGTGTAGAGTTGTTTTTG 36 pGAPD P19_FOR_pGAPD1560Pick up CACTAGTATACCCGGGACATCCGATGTGTAGTTA 37 pGAPD P37_FOR_checkGFPColony GGTTGAATTAGATGGTGATGTTAATG 38 PCR primer P35_REV_checkcasINColony GAGCTCAAGACGCGTTTACTCAATGC 39 PCR primer P31_REV_cassetteAmplification GCGTCAGATTAGCCTCCGACATAG 40 cassette Bold charactersrepresent non-binding extensions.

TABLE 7 Primers used for creating the amylase expressioncassette and control of amylase transformants. Seq ID Name FeatureSequence No. sAmyAOfw Control GGTAGCAGCGTTGATTACTC 41 cassetteconstruction sAmyAOrv Control ATCTGTGCCCTTACGCATAG 42 cassetteconstruction sAmyAOfw checking GGTAGCAGCGTTGATTACTC 43 amy coding regionintegrity sAmyAOrv checking ATCTGTGCCCTTACGCATAG 44 amy coding regionintegrity sAmyAOfw2 checking CCGACAGCGAGCTGTACAAG 45 integrationcassette P35 checking GAGCTCAAGACGCGTTTACT- 46 integration CAATGCcassette

TABLE 8Primers used for creating the PHA expression cassette. All primers were obtained from Sigma Genosys. Seq ID Name Feature Sequence No.P53_FOR_downCYP_extSpeI2 cloning TTACTAGTGTTTCTTAGCCTCCCATG 47downstream GAAGAAACG region cyp52M1 P54_REV_downCYP_extNotI2 cloningAATTGGCCTTGCGGCCGCGGTGTC 48 downstream GACTCGCCAAATTCCATC region cyp52M1P55_FOR_upCYP_extNheI Amplification GTTGCTAGCTCTCGGCAGATTTCCT 49synthetic TG construct P58_REV_PHAC1 + tCYP_extEcorI amplificationAGAATTCGTCGGTTAAACGCACTCC 50 synthetic TTCA construct P63_FOR_cassPHAC1amplification CTCTCGGCAGATTTCCTTGTG 51 PHA expression cassetteP64_REV_cassPHAC1 Amplification GGTGTCGACTCGCCAAATTC 52 PHA expressioncassette P9_FOR_seqQCSapI_URA3down checking GCACACTTCAACCTTCCTAC 53integration cassette A21TotRev checking GCTCTTGTTCGGTACTCTTATTG 54integration cassette UDPGTA1R Sequencing CCTACCTCTCTTCCCTGATCT 55 primer

TABLE 9Primers used for creation of the UGT1 and CepB expression cassettes. All primers were obtained from Sigma Genosys Seq ID Name FeatureSequence No. MDR 505Rev High fidelity forward 5′ CCTCGCCACCACCTAGTTTG 3′56 primer Ugt1 High fidelity reverse 5′ 57 1737Rev_SalI primerGATCGTCGACTCAAAAGAGGCGGACTTCTGCC 3′ CepB High fidelity reverse 5′GATCGTCGACTCATCCCATTATAACAACAC 58 2355Rev_SalI primer 3′ TK F_SalIHigh fidelity forward 5′ AATTGTCGACGGGAGATGGGGGAGGCTAAC 59 primer 3′TK R_SalI High fidelity reverse 5′ GAGTACGCGTTGAACAAACGACCCAACACC 60primer 3′ URA3 677F_MluI High fidelity forward 5′GAGAACGCGTGATAGTACAGGCTTTGC 3′ 61 primer PCepBT214 High fidelity reverse5′ CATAACGCGTTTCTGCTCTCAACACCGAG 3′ 62 R_MluI primer GTII +1296RevHigh fidelity reverse 5′ AGAAGCTAATTCACTAATTGCCGAC 3′ 63 primer

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What is claimed is:
 1. A method of producing one or more compounds byfermentation comprising culturing a modified Candida bombicola strain toproduce the one or more compounds, wherein said Candida bombicola strainhas, compared to the unmodified wild-type strain: a) at least onedeletion in an endogenous gene encoding an enzyme selected from thegroup consisting of the cytochrome P450 monooxygenase CYP52M1 having thesequence of SEQ ID NO: 67, the glucosyltransferase UGTA1 having sequenceof SEQ ID NO: 2, and the glucosyltransferase UGTB1 having the sequenceof SEQ ID NO: 4, and b) a reduction in its capability of producingsophorolipids of at least 75%, and wherein said sophorolipids areconstituted of the sugar sophorose attached to a C₁₆, C₁₈, C₂₂ or C₂₄hydroxylated fatty acid.
 2. The method according to claim 1 wherein saidone or more compounds are selected from the group consisting ofrecombinant proteins, beta-hydroxy fatty acids andpolyhydroxyalkanoates, dicarboxylic acids, polyunsaturated fatty acids,hydroxylated fatty acids, glycolipids, glucolipids, trehaloselipidsrhamnolipids, sophorolipids with a fatty acid tail ranging from 10 to 15carbon atoms, sophorolipids with a fatty acid tail of 17 carbon atoms,sophorolipids with a fatty acid tail ranging from 19 to 25 carbon atoms,sophorolipids with branched fatty acid tail, sophorolipids with multiplehydroxylated fatty acid tails, fully lactonized sophorolipids and fullyacidic sophorolipids, rhamnose, sophorose, polyketide antibiotics, fattyacid based lactonic structures, organic acids, oleagenious compounds,hydrophobic compounds, squaleen, vitamin D, resveratrol, steroids, andcarotenoides.
 3. The method according to claim 1, wherein said reductionin its capability of producing sophorolipids is 100%.
 4. The methodaccording to claim 1, wherein said Candida bombicola is the strainCandida bombicola ATCC 22214.